Antibodies to bradykinin b1 receptor ligands

ABSTRACT

The invention provides antibodies that specifically bind to Kallidin or des-Arg10-Kallidin. The invention also provides pharmaceutical compositions, as well as nucleic acids encoding anti-Kallidin or des-Arg10-Kallidin antibodies, recombinant expression vectors and host cells for making such antibodies, or fragments thereof. Methods of using antibodies of the invention to modulate Kallidin or des-Arg10-Kallidin activity or detect Kallidin or des-Arg10-Kallidin or, either in vitro or in vivo, are also provided by the invention. The invention further provides methods of making antibodies that specifically bind to des-Arg 9 -Bradykinin and des-Arg 10 -Kallidin-like peptide.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/616,845, filed Mar. 28, 2012, and French Patent Application Number1350953, filed Feb. 4, 2013. The contents of these applications are eachhereby incorporated by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 15, 2013, isnamed 543895_SA9-029PC_Seq_List.txt and is 89,291 bytes in size.

BACKGROUND OF THE INVENTION

The bradykinin B1 receptor has been implicated in pathogenesis ofinflammatory disease and chronic pain. By modulating tissue inflammationand renal fibrosis, the B1 receptor has also been associated withpathogenesis of acute kidney injury as well as chronic kidney diseaseswhich are the main causes of end-stage renal failure.

In humans, the major agonists of the bradykinin B1 receptor are thekinins. Kinins are bioactive peptides produced from the proteolyticcleavage of kininogen proteins. The major kinin agonists of bradykininB1 receptor are the decapeptide Kallidin, and the nonapeptidedes-Arg₁₀-Kallidin (formed by the proteolytic cleavage the c-terminalarginine form Kallidin). Therefore, agents that can inhibit the bindingof Kallidin and des-Arg₁₀-Kallidin to the bradykinin B1 receptor havethe potential to treat or prevent bradykinin B1 receptor-mediatedpathologies.

Accordingly, there is a need in the art for novel agents that inhibitthe binding of Kallidin and des-Arg₁₀-Kallidin to the bradykinin B1receptor for use in the treatment of bradykinin B1 receptor-mediatedhuman pathologies.

SUMMARY OF THE INVENTION

The present invention provides antibodies, or antigen binding fragmentsthereof, that specifically bind Kallidin and des-Arg₁₀-Kallidin andprevent binding to the bradykinin B1 receptor. Such antibodies areparticularly useful for treating Kallidin anddes-Arg₁₀-Kallidin-associated diseases or disorders (e.g., pain orfibrosis). The invention also provides pharmaceutical compositions, aswell as nucleic acids encoding anti-Kallidin and des-Arg₁₀-Kallidinantibodies, recombinant expression vectors and host cells for makingsuch antibodies, or fragments thereof. Methods of using antibodies, orfragments thereof, of the invention to detect Kallidin anddes-Arg₁₀-Kallidin or to modulate Kallidin and des-Arg₁₀-Kallidinactivity, either in vitro or in vivo, are also encompassed by theinvention. The invention also provides methods of making antibodies thatspecifically bind to des-Arg₉-Bradykinin and des-Arg₁₀-Kallidin-likepeptide.

Accordingly, in one aspect the invention provides an isolated monoclonalantibody or antigen binding fragment thereof that:

a) specifically binds to Kallidin or des-Arg₁₀-Kallidin but not toBradykinin or des-Arg₉-Bradykinin;

b) specifically binds to Kallidin or des-Arg₁₀-Kallidin with a KD ofless than 1×10⁻¹⁰ M;

c) specifically binds to Kallidin or des-Arg₁₀-Kallidin with a K_(off)of less than 1×10⁴ s⁻¹; or

d) specifically binds to Kallidin or des-Arg₁₀-Kallidin and inhibitsbinding to the bradykinin B1 receptor.

In one embodiment, the antibody or antigen binding fragment thereofbinds to the N-terminal Lysine residue of Kallidin ordes-Arg₁₀-Kallidin.

In another embodiment, the antibody or antigen binding fragment thereofinhibits the binding of Kallidin or des-Arg₁₀-Kallidin to a bradykinin-1receptor.

In another embodiment, the antibody or antigen binding fragment thereofbinds specifically to mouse Kallidin-like peptide (KLP).

In another embodiment, the antibody or antigen binding fragment thereofcomprises a heavy chain variable domain comprising an HCDR3 amino acidsequence selected from the group consisting of:

a) SEQ ID NO: 7 [X₁Y X₂ X₃D X₄HAM X₅Y], wherein

X₁ is Y, F or H,

X₂ is R, D, A, V, L, I, M, F, Y or W,

X₃ is Y, F, W or H,

X₄ is D, E or Y, and,

X₅ is D or E;

b) SEQ ID NO: 63 [X₁ EYDGX₂YX₃X₄LDX₅], wherein

X₁ is W or F,

X₂ is N or no amino acid;

X₃ is Y or S,

X₄ is D or P, and

X₅ is For Y;

c) SEQ ID NO: 13;

d) SEQ ID NO: 32;

e) SEQ ID NO: 40;

f) SEQ ID NO: 47; and

g) SEQ ID NO: 55.

In another embodiment, the antibody or antigen binding fragment thereofcomprises an HCDR2 amino acid sequence selected from the groupconsisting of:

a) SEQ ID NO: 8 [YFX₁ PX₂NGNTGYNQKFRG], wherein

X₁ is D, R, A, V, L, I, M, F, Y or W, and

X₂ is Y, D, E, N, or Q;

b) SEQ ID NO: 64 [WX₁DPENGDX₂X₃YAPKFQG], wherein

X₁ is I, or V,

X₂ is T, or S, and

X₃ is G, or D;

c) SEQ ID NO: 14

d) SEQ ID NO: 33;

e) SEQ ID NO: 41;

f) SEQ ID NO: 48; and

g) SEQ ID NO: 56.

In another embodiment, the antibody or antigen binding fragment thereofcomprises an HCDR1 amino acid sequence selected from the groupconsisting of:

a) SEQ ID NO: 9 [GYSFTDYX₁IY], In wherein X₁ is N, W or Y;

b) SEQ ID NO: 65 [GFNIKDYYX₁H], wherein X₁ is L, or M;

c) SEQ ID NO: 15;

d) SEQ ID NO: 34;

e) SEQ ID NO: 42;

f) SEQ ID NO: 49; and

g) SEQ ID NO: 57.

In another embodiment, the antibody or antigen binding fragment thereofcomprises a light chain variable domain comprising an LCDR3 amino acidsequence selected from the group consisting of:

a) SEQ ID NO: 10 [QQ X₁ X₂S X₃P X₄T], wherein

X₁ is Y, F or H,

X₂is Y, F, H or W,

X₃ is Y, F, T or H, and,

X₄ is W, Y, F, H or L:

b) SEQ ID NO: 66 [QX₁X₂X₃SX₄PX₅T], wherein

X₁ is Q or N,

X₂ is Y, F, D or H,

X₃ is Y, F, H or W,

X₄ is Y, F, T or H, and

X₅ is W, Y, F, H or L;

c) SEQ ID NO: 69 [X₁QGTHFPYT], wherein X₁ is L or M;

d) SEQ ID NO: 16;

e) SEQ ID NO: 35;

f) SEQ ID NO: 43;

g) SEQ ID NO: 50; and

h) SEQ ID NO: 58.

In another embodiment, the antibody or antigen binding fragment thereofcomprises an LCDR2 amino acid sequence selected from the groupconsisting of:

a) SEQ ID NO: 11 [WASTRX₁], wherein X₁ is E, D, Q or N;

b) SEQ ID NO: 67 [X₁ASTRX₂], wherein

X₁ is W or G, and

X₂ is E, D, Q or N;

c) SEQ ID NO: 17;

d) SEQ ID NO: 36;

e) SEQ ID NO: 51; and

f) SEQ ID NO: 59.

In another embodiment, the antibody or antigen binding fragment thereofcomprises an LCDR1 amino acid sequence selected from the groupconsisting of:

a) SEQ ID NO: 12 [KSSQSLL X₁SSNQKN X₂LA], wherein

X₁ is W, H, Y or F, and

X₂ is H or Y;

b) SEQ ID NO: 68 [KSSQSLLX₁X₂SX₃QX₄NX₅LA], wherein

X₁ is W, H, Y or F,

X₂ is S or G,

X₃ is N or D,

X₄ is K or R,

X₅ is H or Y.

c) SEQ ID NO: 70 [KSSQSLLYSNGX₁TYLN], wherein X₁ is K or E;

b) SEQ ID NO: 18;

c) SEQ ID NO: 37;

d) SEQ ID NO: 44;

e) SEQ ID NO: 52; and

f) SEQ ID NO: 60.

In another embodiment, the antibody or antigen binding fragmentcomprises a light chain variable domain comprising an LCDR3 amino acidsequence selected from the group consisting of:

a) SEQ ID NO: 10 [QQ X₁ X₂S X₃P X₄T], wherein

X₁ is Y, F or H,

X₂ is Y, F, H or W,

X₃ is Y, F, T or H, and,

X₄ is W, Y, F, H or L:

b) SEQ ID NO: 66 [QX₁X₂X₃SX₄PX₅T], wherein

X₁ is Q or N,

X₂ is Y, F, D or H,

X₃ is Y, F, H or W,

X₄ is Y, F, T or H, and

X₅ is W, Y, F, H or L;

c) SEQ ID NO: 69 [X₁QGTHFPYT], wherein X₁ is L or M;

d) SEQ ID NO: 16;

e) SEQ ID NO: 35;

f) SEQ ID NO: 43;

g) SEQ ID NO: 50; and

h) SEQ ID NO: 58.

In another embodiment, the antibody or antigen binding fragment thereofcomprises an LCDR2 amino acid sequence selected from the groupconsisting of:

a) SEQ ID NO: 11 [WASTRX₁], wherein X₁ is E, D, Q or N;

b) SEQ ID NO: 67 [X₁ASTRX₂], wherein

X₁ is W or G, and

X₂ is E, D, Q or N;

c) SEQ ID NO: 17;

d) SEQ ID NO: 36;

e) SEQ ID NO: 51; and

f) SEQ ID NO: 59.

In another embodiment, the antibody or antigen binding fragment thereofcomprises an LCDR1 amino acid sequence selected from the groupconsisting of:

a) SEQ ID NO: 12 [KSSQSLL X₁SSNQKN X₂LA], wherein

X₁ is W, H, Y or F, and

X₂ is H or Y;

b) SEQ ID NO: 68 [KSSQSLLX₁X₂SX₃QX₄NX₅LA], wherein

X₁ is W, H, Y or F,

X₂ is S or G,

X₃ is N or D,

X₄ is K or R,

X₅ is H or Y.

c) SEQ ID NO: 70 [KSSQSLLYSNGX₁TYLN], wherein X₁ is K or E;

b) SEQ ID NO: 18;

c) SEQ ID NO: 37;

d) SEQ ID NO: 44;

e) SEQ ID NO: 52; and

f) SEQ ID NO: 60.

In another embodiment, the antibody or antigen binding fragment thereofcomprises a heavy chain variable region comprising the HCDR3, HCDR2 andHCDR1 region amino sequences set forth in SEQ ID NOs 13, 14, and 15,respectively, and one or more amino acid substitutions at positionsselected from the group consisting of H1, H5, H9, H11, H12, H16, H38,H40, H41, H43, H44, H66, H75, H79, H81, H82A, H83, H87, and H108according to Kabat.

In another embodiment, the antibody or antigen binding fragment thereofcomprises a light chain variable region comprising the LCDR3, LCDR2 andLCDR1 region amino sequences set forth in SEQ ID NOs 16, 17, and 18,respectively, and one or more amino acid substitution at positionsselected from the group consisting of L5, L9, L15, L18, L19, L21, L22,L43, L63, L78, L79, L83, L85, L100 and L104, according to Kabat.

In another embodiment, the antibody or antigen binding fragment thereofcomprises a heavy chain variable region amino acid sequence with atleast 90% identity to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 19, 20, 21, 22, 24, 25, 38, 45, 53, and 61.

In another embodiment, the antibody or antigen binding fragment thereofcomprises a light chain variable domain amino acid sequence with atleast 90% identity to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 26, 27, 28, 29, 29, 30, 31, 39, 46, 54, and62.

In another embodiment, the antibody or antigen binding fragment thereofcomprises a light chain variable region amino acid sequence with atleast 90% identity to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 26, 27, 28, 29, 29, 30, 31, 39, 46, 54, and62.

In another embodiment, the antibody or antigen binding fragment thereofcomprises a heavy chain variable domain comprising an amino acidsequence selected from the group consisting of: SEQ ID NO: 19, 20, 21,22, 24, 25, 38, 45, 53, and 61.

In another embodiment, the antibody or antigen binding fragment thereofcomprises a light chain variable domain amino acid sequence selectedfrom the group consisting of: SEQ ID NO: 26, 27, 28, 29, 29, 30, 31, 39,46, 54, and 62.

In another embodiment, the antibody or antigen binding fragment thereofcomprises an amino acid sequence selected from the group consisting of:SEQ ID NO: 26, 27, 28, 29, 29, 30, 31, 39, 46, 54, and 62.

In another embodiment, the antibody or antigen binding fragment thereofcomprises the heavy chain and light chain variable region amino acidsequences set forth in SEQ ID NO: 19 and 26, SEQ ID NO: 20 and 27, SEQID NO: 21 and 28; SEQ ID NO: 22 and 28; SEQ ID NO: 23 and 29; SEQ ID NO:24 and 30; SEQ ID NO: 25 and 31; SEQ ID NO: 38 and 39, SEQ ID NO: 45 and46, SEQ ID NO: 53 and 54, or SEQ ID NO: 61 and 62, respectively.

In another aspect, the invention provides an antibody, or antigenbinding fragment thereof, that specifically binds to Kallidin anddes-Arg₁₀-Kallidin, wherein the antibody, or antigen binding fragmentthereof, competes for binding to Kallidin and des-Arg₁₀-Kallidin with anantibody comprising the heavy chain and light chain variable regionamino acid sequences set forth in SEQ ID NO: 19 and 26, SEQ ID NO: 38and 39, SEQ ID NO: 45 and 46, SEQ ID NO: 53 and 54, or SEQ ID NO: 61 and62, respectively.

In another aspect, the invention provides an isolated monoclonalantibody or antigen binding fragment thereof that competes for bindingto Kallidin or des-Arg₁₀-Kallidin with the antibody of any one of thepreceding claims, and does not bind to Bradykinin or desArg₉-Bradykinin.

In another aspect, the invention provides an isolated monoclonalantibody or antigen binding fragment thereof that specifically binds toa conformational epitope of kallidin (KD) or desArg10-Kallidin (DAKD)which adopts a Pro4 kink conformation comprising a type II tight turn atProline 4 of the KD or DAKD). In one embodiment, the Pro 4 kinkconformation of KD or DAKD further comprises amino acid repeats of asigmoid shape which align the hydrophobic side chains of the amino acidsin a spatially stacking mode. In another embodiment, the antibody orantigen binding fragment thereof comprises (a) specifically bindsKallidin or des-Arg₁₀-Kallidin but not to Bradykinin ordes-Arg₉-Bradykinin; b) specifically binds to Kallidin ordes-Arg₁₀-Kallidin with a KD of less than 1×10⁻¹⁰ M; c) specificallybinds to Kallidin or des-Arg₁₀-Kallidin with a K_(off) of less than1×10⁴ s⁻¹; or d) specifically binds to Kallidin or des-Arg₁₀-Kallidinand inhibits binding to the bradykinin B1 receptor.

In another aspect, the antibody or antigen binding fragment of theinvention is conjugated to a diagnostic or therapeutic agent.

In another aspect, the invention provides isolated nucleic acid encodingthe amino acid sequence of the antibody, or antigen binding fragmentthereof, of the invention.

In another aspect, the invention provides recombinant expression vectorcomprising the nucleic acid of the invention.

In another aspect, the invention provides a host cell comprising therecombinant expression vector of the invention.

In another aspect, the invention provides a method of producing anantibody that binds specifically to Kallidin and des-Arg₁₀-Kallidin,comprising culturing the host cell of the invention under conditionssuch that an antibody that binds specifically to Kallidin anddes-Arg₁₀-Kallidin is produced by the host cell.

In another aspect, the invention provides a pharmaceutical compositioncomprising the antibody, or antigen binding fragment thereof, of theinvention and one or more pharmaceutically acceptable carriers.

In another aspect, the invention provides a method for treating adisease or disorder Kallidin or des-Arg₁₀-Kallidin-associated disease ordisorder, the method comprising administering to a subject in need ofthereof the pharmaceutical composition of the invention.

In one embodiment, the disease or disorder is chronic pain.

In another aspect, the invention provides a method of generating anantibody that specifically binds to des-Arg₉-Bradykinin anddes-Arg₁₀-Kallidin-like peptide comprising: immunizing an animal with animmunogen comprising a peptide, wherein the peptide consists of theamino acid sequence set forth in SEQ ID No. 11, and wherein the aminoterminal arginine of the peptide is indirectly coupled to a carriermoiety through a linker moiety, such that an antibody that specificallybinds to des-Arg₉-Bradykinin, des-Arg₁₀-Kallidin anddes-Arg₁₀-Kallidin-like peptide is produced by the immune system of theanimal.

In another embodiment, the method further comprises isolating from theanimal, the antibody, a nucleic isolating encoding the antibody, or animmune cell expressing the antibody.

In one embodiment, the carrier moiety is a protein. In anotherembodiment, the protein is Keyhole limpet hemocyanin (KLH). In anotherembodiment, wherein the linker moiety comprises [Gly-Gly-Gly]n, whereinn is at least 1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the results of ELISA assays demonstrating binding of EE1antibody to kinin peptides.

FIG. 2 depicts the results of differential scanning calorimetrymeasurements of antibody F151.

FIG. 3 depicts amino acid sequence alignments of the variable regions ofthe murine and humanized F151 antibody. All identical residues arelisted in the alignment, while homologous residues are identified by “+”sign and non-homologous residues are left blank. Figure discloses SEQ IDNOS 19, 24, 26, and 30, respectively, in order of appearance.

FIG. 4 depicts an electron density map of the antigen binding site ofthe F151antibody/kallidin complex.

FIG. 5 depicts an electron density map of the antigen binding site ofthe F151antibody/des-Arg10-kallidin complex.

FIG. 6 depicts a ribbon and stick representation of the Fv subunit ofF151 bound to kallidin.

FIG. 7 depicts an amino acid sequence alignment of the light chainvariable regions of exemplary murine anti-kallidin antibodies of theinvention. Amino acid residues that interact with kallidin are markedwith asterisks. Figure discloses SEQ ID NOS 134, 125, 124, 123, 126, and131, respectively, in order of appearance.

FIG. 8 depicts an amino acid sequence alignment of the heavy chainvariable regions of exemplary murine anti-kallidin antibodies of theinvention. Amino acid residues that interact with kallidin are markedwith asterisks. Figure discloses SEQ ID NOS 135, 120, 119, 118, 121, and122, respectively, in order of appearance.

FIG. 9 depicts the results of in vivo experiments determining the effectof EE1 antibody on formalin-induced acute inflammatory pain.

FIG. 10 depicts the results of in vivo experiments determining theeffect of EE1 antibody on CFA-induced mechanical hypersensitivity.

FIG. 11 depicts the results of in vivo experiments determining theeffect of EE1 antibody on CFA-induced thermal hypersensitivity.

FIG. 12 depicts the results of in vivo experiments determining theeffect of EE1 antibody on CCI-induced mechanical hypersensitivity.

FIG. 13 depicts the results of in vivo experiments determining theeffect of EE1 antibody on CCI-induced thermal hypersensitivity.

FIG. 14 depicts schematic maps of VL and VH expression constructs forgenerating humanized F151 variant HC3a/LC3a with the restriction DNAendonuclease sites presented as deduced sequences in bold andunderlined. Panel A depicts the light chain and Panel B depicts theheavy chain. Figure discloses SEQ ID NOS 30, 24, and 136, respectively,in order of appearance.

FIG. 15 depicts an alignment of the F151 heavy chain (A) and light chain(B) amino acid sequences with the closest human germline amino acidsequences.

FIG. 16 depicts an alignment of the F151 heavy chain (A) and light chain(B) with a heavy chain locus (1-08 & 1-18) and light chain (V IV-B3)locus of the VH1 sub-family. CDR regions and Vernier regions areindicated in boldface and humanizing mutations are underlined.

FIG. 17 depicts (A) the secondary and (B) tertiary structure of the mainchain polypeptide backbone conformation of kallidin (KD) as bound toF151 antibody which comprises a type II tight turn at Proline 4 (C).Figure discloses SEQ ID NOS 2, 2, and 133, respectively, in order ofappearance.

DETAILED DESCRIPTION

The present invention provides antibodies that specifically bind toKallidin and des-Arg₁₀-Kallidin and prevent binding to the bradykinin B1receptor. Such antibodies are particularly useful for treating Kallidinand des-Arg₁₀-Kallidin -associated disease or disorders (e.g., pain).The invention also provides pharmaceutical compositions, as well asnucleic acids encoding anti-Kallidin and des-Arg₁₀-Kallidin antibodies,recombinant expression vectors and host cells for making suchantibodies, or fragments thereof. Methods of using antibodies of theinvention to detect Kallidin and des-Arg₁₀-Kallidin or to modulateKallidin and des-Arg₁₀-Kallidin activity, either in vitro or in vivo,are also encompassed by the invention.

I. Definitions

In order that the present invention may be more readily understood,certain terms are first defined.

As used herein, the term “Kallidin” refers to a peptide comprising orconsisting of the amino acid sequence KRPPGFSPFR (SEQ ID NO. 1).

As used herein, the term “des-Arg₁₀-Kallidin” refers to a peptidecomprising or consisting of the amino acid sequence KRPPGFSPF (SEQ IDNO. 2).

As used herein, the term “mouse Kallidin” or “Kallidin-like peptide”refers to a peptide comprising or consisting of the amino acid sequenceRRPPGFSPFR (SEQ ID NO. 3)

As used herein, the term “mouse des-Arg₁₀-Kallidin” or“des-Arg₁₀Kallidin-like peptide” refers to a peptide comprising orconsisting of the amino acid sequence RRPPGFSPF (SEQ ID NO. 4).

As used herein, the term “Bradykinin” refers to a peptide comprising orconsisting of the amino acid sequence RPPGFSPFR (SEQ ID NO. 5).

As used herein, the term “des-Arg₉-Bradykinin” refers to a peptidecomprising or consisting of the amino acid sequence RPPGFSPF (SEQ ID NO.6).

As used herein, the term “antibody” refers to immunoglobulin moleculescomprising four polypeptide chains, two heavy (H) chains and two light(L) chains inter-connected by disulfide bonds, as well as multimersthereof (e.g., IgM). Each heavy chain comprises a heavy chain variableregion (abbreviated V_(H) or VH)and a heavy chain constant region (C_(H)or CH). The heavy chain constant region comprises three domains, C_(H)1,C_(H)2 and C_(H)3. Each light chain comprises a light chain variableregion (abbreviated V_(L)) and a light chain constant region (C_(L) orCL). The light chain constant region comprises one domain (C_(L)1). TheV_(H) and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDRs),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDRs and fourFRs, arranged from amino-terminus to carboxy-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

As used herein, the term “antigen-binding fragment” of an antibodyincludes any naturally occurring, enzymatically obtainable, synthetic,or genetically engineered polypeptide or glycoprotein that specificallybinds an antigen to form a complex. Antigen-binding fragments of anantibody may be derived, e.g., from full antibody molecules using anysuitable standard techniques such as proteolytic digestion orrecombinant genetic engineering techniques involving the manipulationand expression of DNA encoding antibody variable and optionally constantdomains. Non-limiting examples of antigen-binding portions include: (i)Fab fragments; (ii) F(ab′)₂ fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR)). Other engineered molecules,such as diabodies, triabodies, tetrabodies and minibodies, are alsoencompassed within the expression “antigen-binding fragment.”

As used herein, the term “CDR” or “complementarity determining region”means the noncontiguous antigen combining sites found within thevariable region of both heavy and light chain polypeptides. Theseparticular regions have been described by Kabat et al., J. Biol. Chem.252, 6609-6616 (1977) and Kabat et al., Sequences of protein ofimmunological interest. (1991), and by Chothia et al., J. Mol. Biol.196:901-917 (1987) and by MacCallum et al., J. Mol. Biol. 262:732-745(1996) where the definitions include overlapping or subsets of aminoacid residues when compared against each other. The amino acid residueswhich encompass the CDRs as defined by each of the above citedreferences are set forth for comparison. In an embodiment of theinvention, the term “CDR” is a CDR as defined by Kabat, based onsequence comparisons.

As used herein the term “framework (FR) amino acid residues” refers tothose amino acids in the framework region of an Ig chain. The term“framework region” or “FR region” as used herein, includes the aminoacid residues that are part of the variable region, but are not part ofthe CDRs (e.g., using the Kabat definition of CDRs). Therefore, avariable region framework is between about 100-120 amino acids in lengthbut includes only those amino acids outside of the CDRs.

As used herein, the term “specifically binds to” refers to the abilityof an antibody or an antigen-binding fragment thereof to bind to anantigen with an Kd of at least about 1×10⁻⁶ M, 1×10⁻⁷ M, 1×10⁻⁸ M,1×10⁻⁹ M, 1×10⁻¹⁰ M, 1×10⁻¹¹ M, 1×10⁻¹² M, or more. The term alsoencompasses refers to the ability of an antibody or an antigen-bindingfragment thereof to bind to an antigen with an affinity that is at leasttwo-fold greater than its affinity for a nonspecific antigen. It shallbe understood, however, that an antibody, or an antigen-binding fragmentthereof, is capable of specifically binding to two or more antigenswhich are related in sequence (e.g., Kallidin or des-Arg10-Kallidin andmouse Kallidin or des-Arg10-Kallidin).

As used herein, the term “antigen” refers to the binding site or epitoperecognized by an antibody or antigen binding fragment thereof.

As used herein, the term “vector” is intended to refer to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. The terms, “plasmid” and “vector” may be usedinterchangeably. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

As used herein, the term “host cell” is intended to refer to a cell intowhich a recombinant expression vector has been introduced. It should beunderstood that this term is intended to refer not only to theparticular subject cell but to the progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term “host cell” as used herein.

As used herein, the term “treat,” “treating,” and “treatment” refer totherapeutic or preventative measures described herein. The methods of“treatment” employ administration to a subject, an antibody or antigenbinding fragment of the present invention, for example, a subject havinga Kallidin and des-Arg₁₀-Kallidin-associated disease or disorder (e.g.an inflammatory disease) or predisposed to having such a disease ordisorder, in order to prevent, cure, delay, reduce the severity of, orameliorate one or more symptoms of the disease or disorder or recurringdisease or disorder, or in order to prolong the survival of a subjectbeyond that expected in the absence of such treatment.

As used herein, the term “Kallidin or des-Arg₁₀-Kallidin -associateddisease or disorder” includes disease states and/or symptoms associatedwith a disease state, where altered levels or activity of Kallidin ordes-Arg₁₀-Kallidin are found. Exemplary Kallidin ordes-Arg₁₀-Kallidin-associated diseases or disorders include, but are notlimited to, pain and fibrosis.

As used herein, the term “effective amount” refers to that amount of anantibody or an antigen binding fragment thereof that binds Kallidin ordes-Arg₁₀-Kallidin, which is sufficient to effect treatment, prognosisor diagnosis of a Kallidin or des-Arg₁₀-Kallidin-associated disease ordisorder, as described herein, when administered to a subject. Atherapeutically effective amount will vary depending upon the subjectand disease condition being treated, the weight and age of the subject,the severity of the disease condition, the manner of administration andthe like, which can readily be determined by one of ordinary skill inthe art. The dosages for administration can range from, for example,about 1 ng to about 10,000 mg, about 1 ug to about 5,000 mg, about 1 mgto about 1,000 mg, about 10 mg to about 100 mg, of an antibody orantigen binding fragment thereof, according to the invention. Dosageregiments may be adjusted to provide the optimum therapeutic response.An effective amount is also one in which any toxic or detrimentaleffects (i.e., side effects) of an antibody or antigen binding fragmentthereof are minimized or outweighed by the beneficial effects.

As used herein, the term “subject” includes any human or non-humananimal.

As used herein, the term “epitope” refers to an antigenic determinantthat interacts with a specific antigen binding site in the variableregion of an antibody molecule known as a paratope. A single antigen mayhave more than one epitope. Thus, different antibodies may bind todifferent areas on an antigen and may have different biological effects.Epitopes may be either conformational or linear. A conformationalepitope is produced by spatially juxtaposed amino acids from differentsegments of the linear polypeptide chain. A linear epitope is oneproduced by adjacent amino acid residues in a polypeptide chain.

It is noted here that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and the include plural referenceunless the context clearly dictates otherwise.

II. Anti-Kallidin or des-Arg₁₀-Kallidin Antibodies

In one aspect the invention provides antibodies, or antigen bindingfragments thereof, that specifically bind to Kallidin ordes-Arg₁₀-Kallidin. Exemplary VH, VL and CDR amino acid sequences of theantibodies of the invention are set forth in Table 1.

TABLE 1 VH, VL and CDR amino acid sequences ofexemplary anti-Kallidin or des-Arg₁₀-Kallidin antibodies. SEQ ID Antibody Clone Sequence NO. F151 HCDR3 X₁YX₂X₃DX₄HAMX₅Y  7 consensuswhere: X₁ is Y, F or H; X₂ is R, D,A, V, L, I, M, F, Y or W;X₃ is Y, F,W or H; X₄ is D, E or Y; and X₅ is D or E. F151 HCDR2YFX₁PX₂NGNTGYNQKFRG  8 consensus where:X₁ is D, R, A, V, L, I, M, F, Y or W; and X₂ is Y, D, E, N, or Q.F151 HCDR1 GYSFTDYX₁IY  9 consensus Where X₁ is N, W or Y. F151 LCDR3QQX₁X₂SX₃PX₄T 10 consensus where: X₁ is Y, F or H; X₂ is Y, F, H or W;X₃ is Y, F, T or H;  and X₄ is W, Y, F, H or L. F151 LCDR2 WASTRX₁ 11consensus where X₁ is E, D, Q or N. F151 LCDR1 KSSQSLLX₁SSNQKNX₂LA 12consensus where: X₁ is W, H, Y or F; and X₂ is H or Y. F151 HCDR3YYRYDDHAMDY 13 F151 HCDR2 YFDPYNGNTGYNQKFRG 14 F151 HCDR1 GYSFTDYNIY 15F151 LCDR3 QQYYSYPWT 16 F151 LCDR2 WASTRES 17 F151 LCDR1KSSQSLLYSSNQKNYLA 18 F151 VH EIQLQQSGPELVKPGTSVKVSCKASGYSFTDYNIYWVKQ 19SHGKSLEWIGYFDPYNGNTGYNQKFRGKATLTVDKSSSTAFMHLSSLTSDDSAVYYCANYYRYDDHAMDYWGQGTSVT VSS F151EIQLVQSGPEVKKPGASVKVSCKASGYSFTDYNIYWVKQ 20 Humanized HC1SPGKSLEWIGYFDPYNGNTGYNQKFRGKATLTVDKSSSTAFMHLSSLTSEDSAVYYCANYYRYDDHAMDYWGQGTSVT VSS F151QIQLVQSGAEVKKPGASVKVSCKASGYSFTDYNIYWVKQ 21 Humanized HC2aSPGKGLEWIGYFDPYNGNTGYNQKFRGKATLTVDKSSSTAYMHLSSLTSEESAVYYCANYYRYDDHAMDYWGQGTSVT VSS F151QIQLVQSGAEVKKPGASVKVSCKASGYSFTDYNIYWVKQ 22 Humanized HC2bSPGKGLEWIGYFDPYNGNTGYNEKFRGKATLTVDKSSSTAYMHLSSLTSEESAVYYCANYYRYDDHAMDYWGQGTSVT VSS F151QIQLVQSGAEVKKPGASVKVSCKASGYSFTDYNIYWVKQ 23 Humanized HC2cSPGKGLEWIGYFDPYNGNTGYNQKFRGKATLTVDKSSSTAYMHLSSKTSEESAVYYCANYYRYDDHAMDYWGQGTSVT VSS F151QIQLVQSGAEVKKPGASVKVSCKASGYSFTDYNIYWVRQ 24 Humanized HC3aAPGQGLEWIGYFDPYNGNTGYNQKFRGRATLTVDKSTSTAYMELRSLRSDDTAVYYCANYYRYDDHAMDYWGQGTLVT VSS F151QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYNIYWVRQ 25 Humanized HC3bAPGQGLEWMGYFDPYNGNTGYNQKFRGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCANYYRYDDHAMDYWGQGTLVT VSS F151 VLDIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYL 26AWYQQKPGQSPKPLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAIYYCQQYYSYPWTFGGGTKLEIK F151DIVMSQSPSSLAASVGDRVTMSCKSSQSLLYSSNQKNYL 27 Humanized LC1AWYQQKPGKSPKPLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAIYYCQQYYSYPWTFGGGTKLEIK F151DIVMTQSPSSLSASVGDRVTISCKSSQSLLYSSNQKNYL 28 Humanized LC2aAWYQQKPGKSPKPLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDLATYYCQQYYSYPWTFGGGTKLEIK F151DIVMTQSPSSLSASVGDRVTISCKSSQSLLYSSNQKNYL 29 Humanized LC2bAWYQQKPGKSPKPLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDKATYYCQQYYSYPWTFGGGTKLEIK F151DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNYL 30 Humanized LC3aAWYQQKPGQPPKPLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPWTFGQGTKVEIK F151DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNYL 31 Humanized LC3bAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPWTFGQGTKVEIK B21 HCDR3 WEYDGYYDLDY 32 B21 HCDR2WIDPENGDTGYARKFQG 33 B21 HCDR1 GFNIKDYYLH 34 B21 LCDR3 LQGTHFPYT 35B21 LCDR2 LVSKLDS 36 B21 LCDR1 KSSQSLLYSNGKTYLN 37 B21 VHEVQLQQSGAELVRSGASVKLSCTASGFNIKDYYLHWVKQ 38RPEQGLEWIGWIDPENGDTGYARKFQGKATMTADTSSNTVYLHLSSLTSEDTAVYYFNAWEYDGYYDLDYWGQGTSVT VSS B21 VLDVVMTQTPLTLSVTIGQPASISCKSSQSLLYSNGKTYLN 39WLLQRPGQSPKRLTYLVSKLDSGVPDRFTGSGSGTDFTLKIIRVEAEDLGVYYCLQGTHEPYTFGGGTKLEIK C63 HCDR3 EDYGGDY 40 C63 HCDR2EIRSKSNNYATHYAESVKG 41 C63 HCDR1 GFTFSNYWMN 42 C63 LCDR3 QQYYSYPYT 43C63 LCDR2 WASTRES 17 C63 LCDR1 KSSQSLLYSSDQRNYLA 44 C63 VHEVKLEESGGGLVQPGGSMKLSCVASGFTFSNYWMNWVR 45QSPEKGLEWVAEIRSKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCIGEDYGGDYWGQGTSV TVSS C63 VLDIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSDQRNYL 46AWYQQRSGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPYTFGGGTKLEIK I22 HCDR3 FEYDGNYSPLDF 47 I22 HCDR2WVDPENGDSDYAPKFQ 48 I22 HCDR1 GFNIKDYYMH 49 I22 LCDR3 QNDHSYPLT 50I22 LCDR2 GASTRES 51 I22 LCDR1 KSSQSLLNSGNQKNYLA 52 I22 VHEVQLQQSGAELVRSGASVKLSCTASGFNIKDYYMHWVKQ 53RPEQGLEWIGWVDPENGDSDYAPKFQGKATMTADTSSNTVYLQFSSLTSEDTAVYYCNAFEYDGNYSPLDFWGQGTSV TVSS I22 VLDIVMTQSPSSLSVSAGEKVTMSCKSSQSLLNSGNQKNYL 54AWYQQKPGQPPKLLIYGASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDHSYPLTFGAGTKLELK I54 HCDR3 FEYDGNYSPLDF 55 I54 HCDR2WVDPENGDSDYAPKFQG 56 I54 HCDR1 GFNIKDYYMH 57 I54 LCDR3 MQGTHFPYT 58I54 LCDR2 LVSKLDS 59 I54 LCDR1 KSSQSLLYSNGETYLN 60 I54 VHEVQLQQSGAELVRSGASVKLSCTASGFNIKDYYMHWVKQ 61RPEQGLEWIGWVDPENGDSDYAPKFQGKATMTADTSSNTVYLQFSSLTSEDTAVYYCNAFEYDGNYSPLDFWGQGTSV TVSS I54 VLDVVMTQTPLTLSVPIGQPASISCKSSQSLLYSNGETYLN 62WLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSRSGTDFTLKISRVESEDLGVYYCMQGTHFPYTFGGGTKLEIK B21/I22/I54 X₁EYDGX₂YX₃X₄LDX₅ 63HCDR3 consensus where: X₁ is W or F; X₂ is N or no amino acid;X₃ is Y or S; X₄ is D or P; and X₅ is F or Y. B21/I22/I54WX₁DPENGDX₂X₃YAPKFQG 64 HCDR2 consensus where: X₁ is I, or V;X₂ is T, or S; and X₃ is G, or D. B21/I22/I54 GFNIKDYYX₁H 65HCDR1 consensus where X₁ is L, or M. F151/C63/I22 QX₁X₂X₃SX₄PX₅T 66LCDR3 consensus where: X₁ is Q or N; X₂ is Y, F, D or H;X₃ is Y, F, H or W; X₄ is Y, F, T or H; and X₅ is W, Y, F, H or L.F151/C63/I22 X₁ASTRX₂ 67 LCDR2 consensus where: X₁ is W or G;  andX₂ is E, D, Q or N F151/C63/I22 KSSQSLLX₁X₂SX₃QX₄NX₅LA 68LCDR1 consensus where: X₁ is W, H, Y or F; X₂ is S or G; X₃ is N or D;X₄ is K or R; X₅ is H or Y. B21/I54 LCDR3 X₁QGTHFPYT 69 consensus where:X₁ is L or M; B21/I54 LCDR2 LVSKLDS 36 both identical B21/I54 LCDR1KSSQSLLYSNGX₁TYLN 70 consensus where: X₁ is K or E;

In certain embodiments, the antibody, or antigen binding fragmentthereof, comprises one or more CDR region amino acid sequence selectedfrom the group consisting of SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 32, 33, 34, 35, 36, 37, 40, 41, 42, 43, 44, 47, 48, 49, 50,51 52, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, and70.

In other embodiments, the antibody, or antigen binding fragment thereof,comprises HCDR3, HCDR2 and HCDR1 region amino acid sequences selectedfrom the group consisting of:

a) SEQ ID NO: 7, 8, and 9;

b) SEQ ID NO: 13, 14, and 15;

c) SEQ ID NO: 32, 33, and 34;

d) SEQ ID NO: 40, 41, and 42;

e) SEQ ID NO: 47, 48, and 49;

f) SEQ ID NO: 55, 56, and 57; and

g) SEQ ID NO: 63, 64, and 65, respectively.

In other embodiments, the antibody, or antigen binding fragment thereof,comprises the LCDR3, LCDR2 and LCDR1 region amino acid sequencesselected from the group consisting of:

a) SEQ ID NO: 10, 11, and 12;

b) SEQ ID NO: 16, 17, and 18;

c) SEQ ID NO: 35, 36, and 37;

d) SEQ ID NO: 43, 17, and 44;

e) SEQ ID NO: 50, 51, and 52;

f) SEQ ID NO: 58, 59, and 60;

g) SEQ ID NO: 66, 67, and 68; and

h) SEQ ID NO: 69, 25, and 70, respectively.

In other embodiments, the antibody, or antigen binding fragment thereof,comprises the HCDR3, HCDR2, HCDR1, LCDR3, LCDR2 and LCDR1 region aminoacid sequences selected from the group consisting of:

a) SEQ ID NO: 7, 8, 9, 10, 11, and 12;

b) SEQ ID NO: 13, 14, 15, 16, 17, and 18;

c) SEQ ID NO: 32, 33, 34, 35, 36 and 37;

d) SEQ ID NO: 40, 41, 42, 43, 17, and 44;

e) SEQ ID NO: 47, 48, 49, 50, 51, and 52; and

f) SEQ ID NO: 55, 56, 57, 58, 59, and 60, respectively

In other embodiment, the invention provides humanized antibodies, orantigen binding fragments thereof, comprising one or more CDR regions(or conservatively modified variants thereof) from the murine antibodiesdisclosed herein. Any method of humanization can be employed to generatethe humanized antibodies of the invention. Suitable methods aredisclosed herein and specifically exemplified in Example 4.

In a one particular embodiment, the humanized antibody, or antigenbinding fragment thereof comprises:

a heavy chain variable region comprising the HCDR3, HCDR2 and HCDR1region amino sequences set forth in 13, 14, and 15, respectively, andone or more amino acid substitution at positions selected from the groupconsisting of H1, H5, H9, H11, H12, H16, H38, H40, H41, H43, H44, H66,H75, H79, H81, H82A, H83, H87, and H108; and/or

a light chain variable region comprising the LCDR3, LCDR2 and LCDR1region amino sequences set forth in 16, 17, and 18, respectively, andone or more amino acid substitution at positions selected from the groupconsisting of L5, L9, L15, L18, L19, L21, L22, L43, L63, L78, L79, L83,L85, L100 and L104 (according to the Kabat numbering convention).

In other embodiments, the antibody, or antigen binding fragment thereof,comprises the VH region amino acid sequences set forth in SEQ ID NO: 19,20, 21, 22, 24, 25, 38, 45, 53, and/or 61.

In other embodiments, the antibody, or antigen binding fragment thereof,comprises the VL region amino acid sequences set forth in SEQ ID NO: 26,27, 28, 29, 29, 30, 31, 39, 46, 54, and/or 62.

In other embodiments, the antibody, or antigen binding fragment thereof,comprises the VH and VL region amino acid sequences selected from thegroup consisting of: SEQ ID NO: 19 and 26, SEQ ID NO: 20 and 27, SEQ IDNO: 21 and 28; SEQ ID NO: 22 and 28; SEQ ID NO: 23 and 29; SEQ ID NO: 24and 30; SEQ ID NO: 25 and 31; SEQ ID NO: 38 and 39, SEQ ID NO: 45 and46, SEQ ID NO: 53 and 54, or SEQ ID NO: 61 and 62, respectively.

In certain embodiments, the antibody, or antigen binding fragmentthereof, comprises one or more CDR region amino acid sequence selectedfrom the group consisting of SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 32, 33, 34, 35, 36, 37, 40, 41, 42, 43, 44, 47, 48, 49, 50,51 52, 55, 56, 57, 58, 59 and 60, wherein the one or more CDR regionamino acid sequences comprises at least one or more conservative aminoacid substitutions.

The present invention also encompasses “conservative amino acidsubstitutions” in the CDR amino acid sequences (e.g., SEQ ID NOs: 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 32, 33, 34, 35, 36, 37, 40, 41, 42,43, 44, 47, 48, 49, 50, 51 52, 55, 56, 57, 58, 59 and 60) of theantibodies of the invention, i.e., amino acid sequence modificationswhich do not abrogate the binding of the antibody to the antigen, e.g.,Kallidin or des-Arg10-Kallidin. Conservative amino acid substitutionsinclude the substitution of an amino acid in one class by an amino acidof the same class, where a class is defined by common physicochemicalamino acid side chain properties and high substitution frequencies inhomologous proteins found in nature, as determined, for example, by astandard Dayhoff frequency exchange matrix or BLOSUM matrix. Six generalclasses of amino acid side chains have been categorized and include:Class I (Cys); Class II (Ser, Thr, Pro, Ala, Gly); Class III (Asn, Asp,Gin, Glu); Class IV (His, Arg, Lys); Class V (Ile, Leu, Val, Met); andClass VI (Phe, Tyr, Trp). For example, substitution of an Asp foranother class HI residue such as Asn, Gin, or Glu, is a conservativesubstitution. Thus, a predicted nonessential amino acid residue in ananti-Kallidin or des-Arg10-Kallidin antibody is preferably replaced withanother amino acid residue from the same class. Methods of identifyingamino acid conservative substitutions which do not eliminate antigenbinding are well-known in the art (see, e.g., Brummell et al., Biochem.32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884(1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

In another embodiment, the present invention provides anti-Kallidin ordes-Arg10-Kallidin antibodies, or antigen binding fragment thereof, thatcomprise a VH and/or VL region amino acid sequence with about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%,identity to the VH region amino acid sequence set forth in SEQ ID NO:19, 20, 21, 22, 24, 25, 38, 45, 53, or 61, or the VL region amino acidsequence set forth in SEQ ID NO: 26, 27, 28, 29, 29, 30, 31, 39, 46, 54,or 62, respectively.

In another embodiment, the present invention provides anti-Kallidin ordes-Arg10-Kallidin antibodies that bind to the same epitope and/or crosscompete with an antibody, or antigen binding fragment thereof comprisingthe VH and VL region amino acid sequences set forth in SEQ ID NO: 19 and25, SEQ ID NO: 38 and 39, SEQ ID NO: 45 and 46, SEQ ID NO: 53 and 54, orSEQ ID NO: 61 and 62, respectively. Such antibodies can be identifiedusing routine competition binding assays including, for example, surfaceplasmon resonance (SPR)-based competition assays.

In certain embodiments, the antibodies of the invention bind aconformational epitope of kallidin (KD) or desArg10-Kallidin (DAKD)which adopts a “Pro4 kink” conformation. As depicted in FIG. 17, ahallmark of the “Pro 4 kink” conformation is a type H tight turn in themain chain polypeptide backbone of KD or DAKD at Proline 4. As known tothose of skill in the art, a type H tight turn conformation comprisesthree residues (X1-X2-X3) with the carbonyl of residue X1 forming ahydrogen bond with the amide N of residue X3, which is typically aglycine (see Richardson J S. “The anatomy and taxonomy of proteinstructure.” Adv Protein Chem. 1981; 34:167-339, which is incorporated byreference herein). Accordingly, in certain embodiments, a type II tightturn conformation is formed by the Pro3-Pro4-Gly5 motif of KD or DADK.In more specific embodiments, the “Pro 4 kink” conformation is furtherdefined by all or substantially all of the remaining amino acids of KD(1-2 and 6-9) or DAKD adopting repeats of a sigmoid shape which alignthe hydrophobic side chains in a spatially stacking mode.

III. Modified Anti-Kallidin or des-Arg₁₀-Kallidin Antibodies

In certain embodiments, anti-Kallidin or des-Arg10-Kallidin antibodiesof the invention may comprise one or more modifications. Modified formsof anti-Kallidin or des-Arg10-Kallidin antibodies of the invention canbe made using any techniques known in the art.

i) Reducing Immunogenicity

In certain embodiments, anti-Kallidin or des-Arg10-Kallidin antibodies,or antigen binding fragments thereof, of the invention are modified toreduce their immunogenicity using art-recognized techniques. Forexample, antibodies, or fragments thereof, can be chimericized,humanized, and/or deimmunized.

In one embodiment, an antibody, or antigen binding fragments thereof, ofthe invention may be chimeric. A chimeric antibody is an antibody inwhich different portions of the antibody are derived from differentanimal species, such as antibodies having a variable region derived froma murine monoclonal antibody and a human immunoglobulin constant region.Methods for producing chimeric antibodies, or fragments thereof, areknown in the art. See, e.g., Morrison, Science 229:1202 (1985); Oi etal., BioTechniques 4:214 (1986); Gillies et al., J. Immunol. Methods125:191-202 (1989); U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397,which are incorporated herein by reference in their entireties.Techniques developed for the production of “chimeric antibodies”(Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984); Neuberger etal., Nature 312:604-608 (1984); Takeda et al., Nature 314:452-454(1985)) may be employed for the synthesis of said molecules. Forexample, a genetic sequence encoding a binding specificity of a mouseanti-Kallidin or des-Arg10-Kallidin antibody molecule may be fusedtogether with a sequence from a human antibody molecule of appropriatebiological activity. As used herein, a chimeric antibody is a moleculein which different portions are derived from different animal species,such as those having a variable region derived from a murine monoclonalantibody and a human immunoglobulin constant region, e.g., humanizedantibodies.

In another embodiment, an antibody, or antigen binding fragment thereof,of the invention is humanized. Humanized antibodies, have a bindingspecificity comprising one or more complementarity determining regions(CDRs) from a non-human antibody and framework regions from a humanantibody molecule. Often, framework residues in the human frameworkregions will be substituted with the corresponding residue from the CDRdonor antibody to alter, preferably improve, antigen binding. Theseframework substitutions are identified by methods well known in the art,e.g., by modeling of the interactions of the CDR and framework residuesto identify framework residues important for antigen binding andsequence comparison to identify unusual framework residues at particularpositions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmannet al., Nature 332:323 (1988), which are incorporated herein byreference in their entireties.) Antibodies can be humanized using avariety of techniques known in the art including, for example,CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos.5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332).

In a particular embodiment, a humanization method is employed that isbased on the impact of the molecular flexibility of the antibody duringand at immune recognition (see WO2009/032661, which is incorporatedherein by reference in its entirety). Protein flexibility is related tothe molecular motion of the protein molecule. Protein flexibility is theability of a whole protein, a part of a protein or a single amino acidresidue to adopt an ensemble of conformations which differ significantlyfrom each other. Information about protein flexibility can be obtainedby performing protein X-ray crystallography experiments (see, forexample, Kundu et al. 2002, Biophys J 83:723-732.), nuclear magneticresonance experiments (see, for example, Freedberg et al., J Am Chem Soc1998, 120(31):7916-7923) or by running molecular dynamics (MD)simulations. An MD simulation of a protein is done on a computer andallows one to determine the motion of all protein atoms over a period oftime by calculating the physical interactions of the atoms with eachother. The output of a MD simulation is the trajectory of the studiedprotein over the period of time of the simulation. The trajectory is anensemble of protein conformations, also called snapshots, which areperiodically sampled over the period of the simulation, e.g. every 1picosecond (ps). It is by analyzing the ensemble of snapshots that onecan quantify the flexibility of the protein amino acid residues. Thus, aflexible residue is one which adopts an ensemble of differentconformations in the context of the polypeptide within which thatresidue resides. MD methods are known in the art, see, e.g., Brooks etal. “Proteins: A Theoretical Perspective of Dynamics, Structure andThermodynamics” (Wiley, New York, 1988). Several software enable MDsimulations, such as Amber (see Case et al. (2005) J Comp Chem26:1668-1688), Charmm (see Brooks et al. (1983) J Comp Chem 4:187-217;and MacKerell et al. (1998) in “The Encyclopedia of ComputationalChemistry” vol. 1:271-177, Schleyer et al., eds. Chichester: John Wiley& Sons) or Impact (see Rizzo et al. J Am Chem Soc; 2000;122(51):12898-12900.)

Most protein complexes share a relatively large and planar buriedsurface and it has been shown that flexibility of binding partnersprovides the origin for their plasticity, enabling them toconformationally adapt to each other (Structure (2000) 8, R137-R142). Assuch, examples of “induced fit” have been shown to play a dominant rolein protein-protein interfaces. In addition, there is a steadilyincreasing body of data showing that proteins actually bind ligands ofdiverse shapes sizes and composition (Protein Science (2002) 11:184-187)and that the conformational diversity appears to be an essentialcomponent of the ability to recognize different partners (Science (2003)299, 1362-1367). Flexible residues are involved in the binding ofprotein-protein partners (Structure (2006) 14, 683-693).

The flexible residues can adopt a variety of conformations that providean ensemble of interaction areas that are likely to be recognized bymemory B cells and to trigger an immunogenic response. Thus, an antibodycan be humanized by modifying a number of residues from the framework sothat the ensemble of conformations and of recognition areas displayed bythe modified antibody resemble as much as possible those adopted by ahuman antibody. That can be achieved by modifying a limited number ofresidues by: (1) building a homology model of the parent mAb and runningan MD simulation; (2) analyzing the flexible residues and identificationof the most flexible residues of a non-human antibody molecule, as wellas identifying residues or motifs likely to be a source of heterogeneityor of degradation reaction; (3) identifying a human antibody whichdisplays the most similar ensemble of recognition areas as the parentantibody; (4) determining the flexible residues to be mutated, residuesor motifs likely to be a source of heterogeneity and degradation arealso mutated; and (5) checking for the presence of known T cell or Bcell epitopes. The flexible residues can be found using an MDcalculation as taught herein using an implicit solvent model, whichaccounts for the interaction of the water solvent with the protein atomsover the period of time of the simulation.

Once the set of flexible residues has been identified within thevariable light and heavy chains, a set of human heavy and light chainvariable region frameworks that closely resemble that of the antibody ofinterest are identified. That can be done, for example, using a BLASTsearch on the set of flexible residues against a database of antibodyhuman germ line sequence. It can also be done by comparing the dynamicsof the parent mAb with the dynamics of a library of germ line canonicalstructures. The CDR residues and neighboring residues are excluded fromthe search to ensure high affinity for the antigen is preserved.Flexible residues then are replaced.

When several human residues show similar homologies, the selection isdriven also by the nature of the residues that are likely to affect thesolution behavior of the humanized antibody. For instance, polarresidues will be preferred in exposed flexible loops over hydrophobicresidues. Residues which are a potential source of instability andheterogeneity are also mutated even if there are found in the CDRs. Thatwill include exposed methionines as sulfoxide formation can result fromoxygen radicals, proteolytic cleavage of acid labile bonds such as thoseof the Asp-Pro dipeptide (Drug Dev Res (2004) 61:137-154), deamidationsites found with an exposed asparagine residue followed by a small aminoacid, such as Gly, Ser, Ala, H is, Asn or Cys (J Chromatog (2006)837:35-43) and N-glycosylation sites, such as the Asn-X-Ser/Thr site.Typically, exposed methionines will be substituted by a Leu, exposedasparagines will be replaced by a glutamine or by an aspartate, or thesubsequent residue will be changed. For the glycosylation site(Asn-X-Ser/Thr), either the Asn or the Ser/Thr residue will be changed.

The resulting composite antibody sequence is checked for the presence ofknown B cell or linear T-cell epitopes. A search is performed, forexample, with the publicly available Immune Epitope Data Base (IEDB)(PLos Biol (2005) 3(3)e91). If a known epitope is found within thecomposite sequence, another set of human sequences is retrieved andsubstituted. Thus, unlike the resurfacing method of U.S. Pat. No.5,639,641, both B-cell-mediated and T-cell-mediated immunogenicresponses are addressed by the method. The method also avoids the issueof loss of activity that is sometimes observed with CDR grafting (U.S.Pat. No. 5,530,101). In addition, stability and solubility issues alsoare considered in the engineering and selection process, resulting in anantibody that is optimized for low immunogenicity, high antigen affinityand improved biophysical properties.

In some embodiments, de-immunization can be used to decrease theimmunogenicity of and antibody, or antigen binding fragment thereof. Asused herein, the term “de-immunization” includes alteration of anantibody, or antigen binding fragment thereof, to modify T cell epitopes(see, e.g., WO9852976A1, WO0034317A2). For example, VH and VL sequencesfrom the starting antibody may be analyzed and a human T cell epitope“map” may be generated from each V region showing the location ofepitopes in relation to complementarity-determining regions (CDRs) andother key residues within the sequence. Individual T cell epitopes fromthe T cell epitope map are analyzed in order to identify alternativeamino acid substitutions with a low risk of altering activity of thefinal antibody. A range of alternative VH and VL sequences are designedcomprising combinations of amino acid substitutions and these sequencesare subsequently incorporated into a range of Kallidin ordes-Arg10-Kallidin-specific antibodies or fragments thereof for use inthe diagnostic and treatment methods disclosed herein, which are thentested for function. Typically, between 12 and 24 variant antibodies aregenerated and tested. Complete heavy and light chain genes comprisingmodified V and human C regions are then cloned into expression vectorsand the subsequent plasmids introduced into cell lines for theproduction of whole antibody. The antibodies are then compared inappropriate biochemical and biological assays, and the optimal variantis identified.

ii) Effector Functions and Fc Modifications

Anti-Kallidin or des-Arg10-Kallidin antibodies of the invention maycomprise an antibody constant region (e.g. an IgG constant region e.g.,a human IgG constant region, e.g., a human IgG1 or IgG4 constant region)which mediates one or more effector functions. For example, binding ofthe Cl component of complement to an antibody constant region mayactivate the complement system. Activation of complement is important inthe opsonisation and lysis of cell pathogens. The activation ofcomplement also stimulates the inflammatory response and may also beinvolved in autoimmune hypersensitivity. Further, antibodies bind toreceptors on various cells via the Fc region, with a Fc receptor bindingsite on the antibody Fc region binding to a Fc receptor (FcR) on a cell.There are a number of Fc receptors which are specific for differentclasses of antibody, including IgG (gamma receptors), IgE (epsilonreceptors), IgA (alpha receptors) and IgM (mu receptors). Binding ofantibody to Fc receptors on cell surfaces triggers a number of importantand diverse biological responses including engulfment and destruction ofantibody-coated particles, clearance of immune complexes, lysis ofantibody-coated target cells by killer cells (called antibody-dependentcell-mediated cytotoxicity, or ADCC), release of inflammatory mediators,placental transfer and control of immunoglobulin production. Inpreferred embodiments, the antibodies, or fragments thereof, of theinvention bind to an Fc-gamma receptor. In alternative embodiments,anti-Kallidin or des-Arg10-Kallidin antibodies of the invention maycomprise a constant region which is devoid of one or more effectorfunctions (e.g., ADCC activity) and/or is unable to bind Fc receptor.

Certain embodiments of the invention include anti-Kallidin ordes-Arg10-Kallidin antibodies in which at least one amino acid in one ormore of the constant region domains has been deleted or otherwisealtered so as to provide desired biochemical characteristics such asreduced or enhanced effector functions, the ability to non-covalentlydimerize, increased ability to localize at the site of a tumor, reducedserum half-life, or increased serum half-life when compared with awhole, unaltered antibody of approximately the same immunogenicity. Forexample, certain antibodies, or fragments thereof, for use in thediagnostic and treatment methods described herein are domain deletedantibodies which comprise a polypeptide chain similar to animmunoglobulin heavy chain, but which lack at least a portion of one ormore heavy chain domains. For instance, in certain antibodies, oneentire domain of the constant region of the modified antibody will bedeleted, for example, all or part of the CH2 domain will be deleted.

In certain other embodiments, anti-Kallidin or des-Arg10-Kallidinantibodies comprise constant regions derived from different antibodyisotypes (e.g., constant regions from two or more of a human IgG1, IgG2,IgG3, or IgG4). In other embodiments, anti-Kallidin ordes-Arg10-Kallidin antibodies comprises a chimeric hinge (i.e., a hingecomprising hinge portions derived from hinge domains of differentantibody isotypes, e.g., an upper hinge domain from an IgG4 molecule andan IgG1 middle hinge domain). In one embodiment, an anti-Kallidin ordes-Arg10-Kallidin antibodies comprises an Fc region or portion thereoffrom a human IgG4 molecule and a Ser228Pro mutation (EU numbering) inthe core hinge region of the molecule.

In certain anti-Kallidin or des-Arg10-Kallidin antibodies, the Fcportion may be mutated to increase or decrease effector function usingtechniques known in the art. For example, the deletion or inactivation(through point mutations or other means) of a constant region domain mayreduce Fc receptor binding of the circulating modified antibody therebyincreasing tumor localization. In other cases it may be that constantregion modifications consistent with the instant invention moderatecomplement binding and thus reduce the serum half life and nonspecificassociation of a conjugated cytotoxin. Yet other modifications of theconstant region may be used to modify disulfide linkages oroligosaccharide moieties that allow for enhanced localization due toincreased antigen specificity or flexibility. The resultingphysiological profile, bioavailability and other biochemical effects ofthe modifications, such as tumor localization, biodistribution and serumhalf-life, may easily be measured and quantified using well knowimmunological techniques without undue experimentation.

In certain embodiments, an Fc domain employed in an antibody of theinvention is an Fc variant. As used herein, the term “Fc variant” refersto an Fc domain having at least one amino acid substitution relative tothe wild-type Fc domain from which said Fc domain is derived. Forexample, wherein the Fc domain is derived from a human IgG1 antibody,the Fc variant of said human IgG1 Fc domain comprises at least one aminoacid substitution relative to said Fc domain.

The amino acid substitution(s) of an Fc variant may be located at anyposition (i.e., any EU convention amino acid position) within the Fcdomain. In one embodiment, the Fc variant comprises a substitution at anamino acid position located in a hinge domain or portion thereof. Inanother embodiment, the Fc variant comprises a substitution at an aminoacid position located in a CH2 domain or portion thereof. In anotherembodiment, the Fc variant comprises a substitution at an amino acidposition located in a CH3 domain or portion thereof. In anotherembodiment, the Fc variant comprises a substitution at an amino acidposition located in a CH4 domain or portion thereof.

The antibodies of the invention may employ any art-recognized Fc variantwhich is known to impart an improvement (e.g., reduction or enhancement)in effector function and/or FcR binding. Said Fc variants may include,for example, any one of the amino acid substitutions disclosed inInternational PCT Publications WO88/07089A1, WO96/14339A1, WO98/05787A1,WO98/23289A1, WO99/51642A1, WO99/58572A1, WO00/09560A2, WO00/32767A1,WO00/42072A2, WO02/44215A2, WO02/060919A2, WO03/074569A2, WO04/016750A2,WO04/029207A2, WO04/035752A2, WO04/063351A2, WO04/074455A2,WO04/099249A2, WO05/040217A2, WO05/070963A1, WO05/077981A2,WO05/092925A2, WO05/123780A2, WO06/019447A1, WO06/047350A2, andWO06/085967A2 or U.S. Pat. Nos. 5,648,260; 5,739,277; 5,834,250;5,869,046; 6,096,871; 6,121,022; 6,194,551; 6,242,195; 6,277,375;6,528,624; 6,538,124; 6,737,056; 6,821,505; 6,998,253; and 7,083,784,each of which is incorporated by reference herein. In one exemplaryembodiment, an antibody of the invention may comprise an Fc variantcomprising an amino acid substitution at EU position 268 (e.g., H268D orH268E). In another exemplary embodiment, an antibody of the inventionmay comprise an amino acid substitution at EU position 239 (e.g., S239Dor S239E) and/or EU position 332 (e.g., 1332D or 13320).

In certain embodiments, an antibody of the invention may comprise an Fcvariant comprising an amino acid substitution which alters theantigen-independent effector functions of the antibody, in particularthe circulating half-life of the antibody. Such antibodies exhibiteither increased or decreased binding to FcRn when compared toantibodies lacking these substitutions, therefore, have an increased ordecreased half-life in serum, respectively. Fc variants with improvedaffinity for FcRn are anticipated to have longer serum half-lives, andsuch molecules have useful applications in methods of treating mammalswhere long half-life of the administered antibody is desired, e.g., totreat a chronic disease or disorder. In contrast, Fc variants withdecreased FcRn binding affinity are expected to have shorter half-lives,and such molecules are also useful, for example, for administration to amammal where a shortened circulation time may be advantageous, e.g. forin vivo diagnostic imaging or in situations where the starting antibodyhas toxic side effects when present in the circulation for prolongedperiods. Fc variants with decreased FcRn binding affinity are also lesslikely to cross the placenta and, thus, are also useful in the treatmentof diseases or disorders in pregnant women. In addition, otherapplications in which reduced FcRn binding affinity may be desiredinclude those applications in which localization the brain, kidney,and/or liver is desired. In one exemplary embodiment, the alteredantibodies of the invention exhibit reduced transport across theepithelium of kidney glomeruli from the vasculature. In anotherembodiment, the altered antibodies of the invention exhibit reducedtransport across the blood brain barrier (BBB) from the brain, into thevascular space. In one embodiment, an antibody with altered FcRn bindingcomprises an Fc domain having one or more amino acid substitutionswithin the “FcRn binding loop” of an Fc domain. The FcRn binding loop iscomprised of amino acid residues 280-299 (according to EU numbering).Exemplary amino acid substitutions which altered FcRn binding activityare disclosed in International PCT Publication No. WO05/047327 which isincorporated by reference herein. In certain exemplary embodiments, theantibodies, or fragments thereof, of the invention comprise an Fc domainhaving one or more of the following substitutions: V284E, H285E, N286D,K290E and S304D (EU numbering).

In other embodiments, antibodies, for use in the diagnostic andtreatment methods described herein have a constant region, e.g., an IgG1or IgG4 heavy chain constant region, which is altered to reduce oreliminate glycosylation. For example, an antibody of the invention mayalso comprise an Fc variant comprising an amino acid substitution whichalters the glycosylation of the antibody. For example, said Fc variantmay have reduced glycosylation (e.g., N- or O-linked glycosylation). Inexemplary embodiments, the Fc variant comprises reduced glycosylation ofthe N-linked glycan normally found at amino acid position 297 (EUnumbering). In another embodiment, the antibody has an amino acidsubstitution near or within a glycosylation motif, for example, anN-linked glycosylation motif that contains the amino acid sequence NXTor NXS. In a particular embodiment, the antibody comprises an Fc variantwith an amino acid substitution at amino acid position 228 or 299 (EUnumbering). In more particular embodiments, the antibody comprises anIgG1 or IgG4 constant region comprising an S228P and a T299A mutation(EU numbering).

Exemplary amino acid substitutions which confer reduce or alteredglycosylation are disclosed in International PCT Publication No.WO05/018572, which is incorporated by reference herein. In preferredembodiments, the antibodies, or fragments thereof, of the invention aremodified to eliminate glycosylation. Such antibodies, or fragmentsthereof, may be referred to as “agly” antibodies, or fragments thereof,(e.g. “agly” antibodies). While not being bound by theory, it isbelieved that “agly” antibodies, or fragments thereof, may have animproved safety and stability profile in vivo. Exemplary aglyantibodies, or fragments thereof, comprise an aglycosylated Fc region ofan IgG4 antibody which is devoid of Fc-effector function therebyeliminating the potential for Fc mediated toxicity to the normal vitalorgans that express Kallidin or des-Arg10-Kallidin. In yet otherembodiments, antibodies, or fragments thereof, of the invention comprisean altered glycan. For example, the antibody may have a reduced numberof fucose residues on an N-glycan at Asn297 of the Fc region, i.e., isafucosylated. In another embodiment, the antibody may have an alterednumber of sialic acid residues on the N-glycan at Asn297 of the Fcregion.

iii) Covalent Attachment

Anti-Kallidin or des-Arg10-Kallidin antibodies of the invention may bemodified, e.g., by the covalent attachment of a molecule to the antibodysuch that covalent attachment does not prevent the antibody fromspecifically binding to its cognate epitope. For example, but not by wayof limitation, the antibodies, or fragments thereof, of the inventionmay be modified by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, etc. Additionally, the derivativemay contain one or more non-classical amino acids.

Antibodies, or fragments thereof, of the invention may further berecombinantly fused to a heterologous polypeptide at the N- orC-terminus or chemically conjugated (including covalent and non-covalentconjugations) to polypeptides or other compositions. For example,anti-Kallidin or des-Arg10-Kallidin antibodies may be recombinantlyfused or conjugated to molecules useful as labels in detection assaysand effector molecules such as heterologous polypeptides, drugs,radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387.

Anti-Kallidin or des-Arg10-Kallidin antibodies may be fused toheterologous polypeptides to increase the in vivo half life or for usein immunoassays using methods known in the art. For example, in oneembodiment, PEG can be conjugated to the anti-Kallidin ordes-Arg10-Kallidin antibodies of the invention to increase theirhalf-life in vivo. Leong, S. R., et al., Cytokine 16:106 (2001); Adv. inDrug Deliv. Rev. 54:531 (2002); or Weir et al., Biochem. Soc.Transactions 30:512 (2002).

Moreover, anti-Kallidin or des-Arg10-Kallidin antibodies of theinvention can be fused to marker sequences, such as a peptide tofacilitate their purification or detection. In preferred embodiments,the marker amino acid sequence is a hexa-histidine peptide (SEQ ID NO:137), such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 EtonAvenue, Chatsworth, Calif., 91311), among others, many of which arecommercially available. As described in Gentz et al., Proc. Natl. Acad.Sci. USA 86:821-824 (1989), for instance, hexa-histidine (SEQ ID NO:137) provides for convenient purification of the fusion protein. Otherpeptide tags useful for purification include, but are not limited to,the “HA” tag, which corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the “flag”tag.

Anti-Kallidin or des-Arg10-Kallidin antibodies of the invention may beused in non-conjugated form or may be conjugated to at least one of avariety of molecules, e.g., to improve the therapeutic properties of themolecule, to facilitate target detection, or for imaging or therapy ofthe patient. Anti-Kallidin or des-Arg10-Kallidin antibodies of theinvention can be labeled or conjugated either before or afterpurification, when purification is performed. In particular,anti-Kallidin or des-Arg10-Kallidin antibodies of the invention may beconjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes,viruses, lipids, biological response modifiers, pharmaceutical agents,or PEG.

The present invention further encompasses anti-Kallidin ordes-Arg10-Kallidin antibodies of the invention conjugated to adiagnostic or therapeutic agent. The anti-Kallidin or des-Arg10-Kallidinantibodies can be used diagnostically to, for example, monitor thedevelopment or progression of a immune cell disorder (e.g., CLL) as partof a clinical testing procedure to, e.g., determine the efficacy of agiven treatment and/or prevention regimen. Detection can be facilitatedby coupling the anti-Kallidin or des-Arg10-Kallidin antibodies to adetectable substance. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, radioactive materials, positronemitting metals using various positron emission tomographies, andnonradioactive paramagnetic metal ions. See, for example, U.S. Pat. No.4,741,900 for metal ions which can be conjugated to antibodies for useas diagnostics according to the present invention. Examples of suitableenzymes include horseradish peroxidase, alkaline phosphatase,.beta.-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin;and examples of suitable radioactive material include 125I, 131I, 111Inor 99Tc.

Anti-Kallidin or des-Arg10-Kallidin antibodies for use in the diagnosticand treatment methods disclosed herein may be conjugated to cytotoxins(such as radioisotopes, cytotoxic drugs, or toxins) therapeutic agents,cytostatic agents, biological toxins, prodrugs, peptides, proteins,enzymes, viruses, lipids, biological response modifiers, pharmaceuticalagents, immunologically active ligands (e.g., lymphokines or otherantibodies wherein the resulting molecule binds to both the neoplasticcell and an effector cell such as a T cell), or PEG.

In another embodiment, an anti-Kallidin or des-Arg10-Kallidin antibodyfor use in the diagnostic and treatment methods disclosed herein can beconjugated to a molecule that decreases tumor cell growth. In otherembodiments, the disclosed compositions may comprise antibodies, orfragments thereof, coupled to drugs or prodrugs. Still other embodimentsof the present invention comprise the use of antibodies, or fragmentsthereof, conjugated to specific biotoxins or their cytotoxic fragmentssuch as ricin, gelonin, Pseudomonas exotoxin or diphtheria toxin. Theselection of which conjugated or unconjugated antibody to use willdepend on the type and stage of cancer, use of adjunct treatment (e.g.,chemotherapy or external radiation) and patient condition. It will beappreciated that one skilled in the art could readily make such aselection in view of the teachings herein.

It will be appreciated that, in previous studies, anti-tumor antibodieslabeled with isotopes have been used successfully to destroy tumor cellsin animal models, and in some cases in humans. Exemplary radioisotopesinclude: 90Y, 125I, 131I, 123I, 111In, 105Rh, 153Sm, 67Cu, 67Ga, 166Ho,177Lu, 186Re and 188Re. The radionuclides act by producing ionizingradiation which causes multiple strand breaks in nuclear DNA, leading tocell death. The isotopes used to produce therapeutic conjugatestypically produce high energy alpha- or beta-particles which have ashort path length. Such radionuclides kill cells to which they are inclose proximity, for example neoplastic cells to which the conjugate hasattached or has entered. They have little or no effect on non-localizedcells. Radionuclides are essentially non-immunogenic.

IV. Expression of Anti-Kallidin or des-Arg10-Kallidin Antibodies, orAntigen Binding Fragments Thereof

Following manipulation of the isolated genetic material to provideanti-Kallidin or des-Arg10-Kallidin antibodies of the invention as setforth above, the genes are typically inserted in an expression vectorfor introduction into host cells that may be used to produce the desiredquantity of the claimed antibodies, or fragments thereof.

The term “vector” or “expression vector” is used herein for the purposesof the specification and claims, to mean vectors used in accordance withthe present invention as a vehicle for introducing into and expressing adesired gene in a cell. As known to those skilled in the art, suchvectors may easily be selected from the group consisting of plasmids,phages, viruses and retroviruses. In general, vectors compatible withthe instant invention will comprise a selection marker, appropriaterestriction sites to facilitate cloning of the desired gene and theability to enter and/or replicate in eukaryotic or prokaryotic cells.

Numerous expression vector systems may be employed for the purposes ofthis invention. For example, one class of vector utilizes DNA elementswhich are derived from animal viruses such as bovine papilloma virus,polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses(RSV, MMTV or MOMLV) or SV40 virus. Others involve the use ofpolycistronic systems with internal ribosome binding sites.Additionally, cells which have integrated the DNA into their chromosomesmay be selected by introducing one or more markers which allow selectionof transfected host cells. The marker may provide for prototrophy to anauxotrophic host, biocide resistance (e.g., antibiotics) or resistanceto heavy metals such as copper. The selectable marker gene can either bedirectly linked to the DNA sequences to be expressed, or introduced intothe same cell by cotransformation. Additional elements may also beneeded for optimal synthesis of mRNA. These elements may include signalsequences, splice signals, as well as transcriptional promoters,enhancers, and termination signals. In particularly preferredembodiments the cloned variable region genes are inserted into anexpression vector along with the heavy and light chain constant regiongenes (preferably human) synthetic as discussed above.

In other preferred embodiments the anti-Kallidin or des-Arg10-Kallidinantibodies, or fragments thereof, of the invention may be expressedusing polycistronic constructs. In such expression systems, multiplegene products of interest such as heavy and light chains of antibodiesmay be produced from a single polycistronic construct. These systemsadvantageously use an internal ribosome entry site (IRES) to providerelatively high levels of polypeptides of the invention in eukaryotichost cells. Compatible IRES sequences are disclosed in U.S. Pat. No.6,193,980, which is incorporated by reference herein. Those skilled inthe art will appreciate that such expression systems may be used toeffectively produce the full range of polypeptides disclosed in theinstant application.

More generally, once a vector or DNA sequence encoding an antibody, orfragment thereof, has been prepared, the expression vector may beintroduced into an appropriate host cell. That is, the host cells may betransformed. Introduction of the plasmid into the host cell can beaccomplished by various techniques well known to those of skill in theart. These include, but are not limited to, transfection (includingelectrophoresis and electroporation), protoplast fusion, calciumphosphate precipitation, cell fusion with enveloped DNA, microinjection,and infection with intact virus. See, Ridgway, A. A. G. “MammalianExpression Vectors” Chapter 24.2, pp. 470-472 Vectors, Rodriguez andDenhardt, Eds. (Butterworths, Boston, Mass. 1988). Most preferably,plasmid introduction into the host is via electroporation. Thetransformed cells are grown under conditions appropriate to theproduction of the light chains and heavy chains, and assayed for heavyand/or light chain protein synthesis. Exemplary assay techniques includeenzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), orflourescence-activated cell sorter analysis (FACS), immunohistochemistryand the like.

As used herein, the term “transformation” shall be used in a broad senseto refer to the introduction of DNA into a recipient host cell thatchanges the genotype and consequently results in a change in therecipient cell.

Along those same lines, “host cells” refers to cells that have beentransformed with vectors constructed using recombinant DNA techniquesand encoding at least one heterologous gene. In descriptions ofprocesses for isolation of polypeptides from recombinant hosts, theterms “cell” and “cell culture” are used interchangeably to denote thesource of antibody unless it is clearly specified otherwise. In otherwords, recovery of polypeptide from the “cells” may mean either fromspun down whole cells, or from the cell culture containing both themedium and the suspended cells.

In one embodiment, the host cell line used for antibody expression is ofmammalian origin; those skilled in the art can determine particular hostcell lines which are best suited for the desired gene product to beexpressed therein. Exemplary host cell lines include, but are notlimited to, DG44 and DUXB11 (Chinese Hamster Ovary lines, DHFR minus),HELA (human cervical carcinoma), CVI (monkey kidney line), COS (aderivative of CVI with SV40 T antigen), R1610 (Chinese hamsterfibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamster kidney line),SP2/O (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI(human lymphocyte), 293 (human kidney). In one embodiment, the cell lineprovides for altered glycosylation, e.g., afucosylation, of the antibodyexpressed therefrom (e.g., PER.C6® (Crucell) or FUT8-knock-out CHO celllines (Potelligent® Cells) (Biowa, Princeton, N.J.)). In one embodimentNS0 cells may be used. CHO cells are particularly preferred. Host celllines are typically available from commercial services, the AmericanTissue Culture Collection or from published literature.

In vitro production allows scale-up to give large amounts of the desiredpolypeptides. Techniques for mammalian cell cultivation under tissueculture conditions are known in the art and include homogeneoussuspension culture, e.g. in an airlift reactor or in a continuousstirrer reactor, or immobilized or entrapped cell culture, e.g. inhollow fibers, microcapsules, on agarose microbeads or ceramiccartridges. If necessary and/or desired, the solutions of polypeptidescan be purified by the customary chromatography methods, for example gelfiltration, ion-exchange chromatography, chromatography overDEAF-cellulose and/or (immuno-)affinity chromatography.

Genes encoding the anti-Kallidin or des-Arg10-Kallidin antibodies, orfragments thereof, of the invention can also be expressed non-mammaliancells such as bacteria or yeast or plant cells. In this regard it willbe appreciated that various unicellular non-mammalian microorganismssuch as bacteria can also be transformed; i.e. those capable of beinggrown in cultures or fermentation. Bacteria, which are susceptible totransformation, include members of the enterobacteriaceae, such asstrains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillussubtilis; Pneumococcus; Streptococcus, and Haemophilus influenzae. Itwill further be appreciated that, when expressed in bacteria, thepolypeptides can become part of inclusion bodies. The polypeptides mustbe isolated, purified and then assembled into functional molecules.

In addition to prokaryotes, eukaryotic microbes may also be used.Saccharomyces cerevisiae, or common baker's yeast, is the most commonlyused among eukaryotic microorganisms although a number of other strainsare commonly available. For expression in Saccharomyces, the plasmidYRp7, for example, (Stinchcomb et al., Nature, 282:39 (1979); Kingsmanet al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)) iscommonly used. This plasmid already contains the TRP1 gene whichprovides a selection marker for a mutant strain of yeast lacking theability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1(Jones, Genetics, 85:12 (1977)). The presence of the trpl lesion as acharacteristic of the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan.

V. Pharmaceutical Formulations and Methods of Administration ofAnti-Kallidin or des-Arg10-Kallidin Antibodies.

In another aspect, the invention provides pharmaceutical compositionscomprising an anti-Kallidin or des-Arg10-Kallidin antibody, or fragmentthereof.

Methods of preparing and administering antibodies, or fragments thereof,of the invention to a subject are well known to or are readilydetermined by those skilled in the art. The route of administration ofthe antibodies, or fragments thereof, of the invention may be oral,parenteral, by inhalation or topical. The term parenteral as used hereinincludes intravenous, intraarterial, intraperitoneal, intramuscular,subcutaneous, rectal or vaginal administration. The intravenous,intraarterial, subcutaneous and intramuscular forms of parenteraladministration are generally preferred. While all these forms ofadministration are clearly contemplated as being within the scope of theinvention, a form for administration would be a solution for injection,in particular for intravenous or intraarterial injection or drip.Usually, a suitable pharmaceutical composition for injection maycomprise a buffer (e.g. acetate, phosphate or citrate buffer), asurfactant (e.g. polysorbate), optionally a stabilizer agent (e.g. humanalbumin), etc. However, in other methods compatible with the teachingsherein, the polypeptides can be delivered directly to the site of theadverse cellular population thereby increasing the exposure of thediseased tissue to the therapeutic agent.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. In the subject invention, pharmaceutically acceptable carriersinclude, but are not limited to, 0.01-0.1 M and preferably 0.05Mphosphate buffer or 0.8% saline. Other common parenteral vehiclesinclude sodium phosphate solutions, Ringer's dextrose, dextrose andsodium chloride, lactated Ringer's, or fixed oils. Intravenous vehiclesinclude fluid and nutrient replenishers, electrolyte replenishers, suchas those based on Ringer's dextrose, and the like. Preservatives andother additives may also be present such as for example, antimicrobials,antioxidants, chelating agents, and inert gases and the like. Moreparticularly, pharmaceutical compositions suitable for injectable useinclude sterile aqueous solutions (where water soluble) or dispersionsand sterile powders for the extemporaneous preparation of sterileinjectable solutions or dispersions. In such cases, the composition mustbe sterile and should be fluid to the extent that easy syringabilityexists. It should be stable under the conditions of manufacture andstorage and will preferably be preserved against the contaminatingaction of microorganisms, such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (e.g., glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), and suitable mixtures thereof. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal and the like. In many cases, it will be preferable to includeisotonic agents, for example, sugars, polyalcohols, such as mannitol,sorbitol, or sodium chloride in the composition. Prolonged absorption ofthe injectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate and gelatin.

In any case, sterile injectable solutions can be prepared byincorporating an active compound (e.g., an antibody by itself or incombination with other active agents) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedherein, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle, which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-drying,which yields a powder of an active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The preparations for injections are processed, filled into containerssuch as ampoules, bags, bottles, syringes or vials, and sealed underaseptic conditions according to methods known in the art. Further, thepreparations may be packaged and sold in the form of a kit such as thosedescribed in co-pending U.S. Ser. No. 09/259,337 and U.S. Ser. No.09/259,338 each of which is incorporated herein by reference. Sucharticles of manufacture will preferably have labels or package insertsindicating that the associated compositions are useful for treating asubject suffering from, or predisposed to autoimmune or neoplasticdisorders.

Effective doses of the stabilized antibodies, or fragments thereof, ofthe present invention, for the treatment of the above describedconditions vary depending upon many different factors, including meansof administration, target site, physiological state of the patient,whether the patient is human or an animal, other medicationsadministered, and whether treatment is prophylactic or therapeutic.Usually, the patient is a human, but non-human mammals includingtransgenic mammals can also be treated. Treatment dosages may betitrated using routine methods known to those of skill in the art tooptimize safety and efficacy.

For passive immunization with an antibody of the invention, the dosagemay range, e.g., from about 0.0001 to 100 mg/kg, and more usually 0.01to 5 mg/kg (e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1mg/kg, 2 mg/kg, etc.), of the host body weight. For example dosages canbe 1 mg/kg body weight or 10 mg/kg body weight or within the range of1-10 mg/kg, preferably at least 1 mg/kg. Doses intermediate in the aboveranges are also intended to be within the scope of the invention.

Subjects can be administered such doses daily, on alternative days,weekly or according to any other schedule determined by empiricalanalysis. An exemplary treatment entails administration in multipledosages over a prolonged period, for example, of at least six months.Additional exemplary treatment regimes entail administration once perevery two weeks or once a month or once every 3 to 6 months. Exemplarydosage schedules include 1-10 mg/kg or 15 mg/kg on consecutive days, 30mg/kg on alternate days or 60 mg/kg weekly. In some methods, two or moremonoclonal antibodies with different binding specificities areadministered simultaneously, in which case the dosage of each antibodyadministered may fall within the ranges indicated.

Antibodies, or fragments thereof, of the invention can be administeredon multiple occasions. Intervals between single dosages can be, e.g.,daily, weekly, monthly or yearly. Intervals can also be irregular asindicated by measuring blood levels of polypeptide or target molecule inthe patient. In some methods, dosage is adjusted to achieve a certainplasma antibody or toxin concentration, e.g., 1-1000 ug/ml or 25-300ug/ml. Alternatively, antibodies, or fragments thereof, can beadministered as a sustained release formulation, in which case lessfrequent administration is required. Dosage and frequency vary dependingon the half-life of the antibody in the patient. In general, humanizedantibodies show the longest half-life, followed by chimeric antibodiesand nonhuman antibodies. In one embodiment, the antibodies, or fragmentsthereof, of the invention can be administered in unconjugated form. Inanother embodiment, the antibodies of the invention can be administeredmultiple times in conjugated form. In still another embodiment, theantibodies, or fragments thereof, of the invention can be administeredin unconjugated form, then in conjugated form, or vise versa.

The dosage and frequency of administration can vary depending on whetherthe treatment is prophylactic or therapeutic. In prophylacticapplications, compositions containing the present antibodies or acocktail thereof are administered to a patient not already in thedisease state to enhance the patients resistance. Such an amount isdefined to be a “prophylactic effective dose.” In this use, the preciseamounts again depend upon the patients state of health and generalimmunity, but generally range from 0.1 to 25 mg per dose, especially 0.5to 2.5 mg per dose. A relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives.

In therapeutic applications, a relatively high dosage (e.g., from about1 to 400 mg/kg of antibody per dose, with dosages of from 5 to 25 mgbeing more commonly used for radioimmunoconjugates and higher doses forcytotoxin-drug conjugated molecules) at relatively short intervals issometimes required until progression of the disease is reduced orterminated, and preferably until the patient shows partial or completeamelioration of symptoms of disease. Thereafter, the patent can beadministered a prophylactic regime.

In one embodiment, a subject can be treated with a nucleic acid moleculeencoding a polypeptide of the invention (e.g., in a vector). Doses fornucleic acids encoding polypeptides range from about 10 ng to 1 g, 100ng to 100 mg, 1 ug to 10 mg, or 30-300 ug DNA per patient. Doses forinfectious viral vectors vary from 10-100, or more, virions per dose.

Therapeutic agents can be administered by parenteral, topical,intravenous, oral, subcutaneous, intraarterial, intracranial,intraperitoneal, intranasal or intramuscular means for prophylacticand/or therapeutic treatment. Intramuscular injection or intravenousinfusion are preferred for administration of a antibody of theinvention. In some methods, therapeutic antibodies, or fragmentsthereof, are injected directly into the cranium. In some methods,antibodies, or fragments thereof, are administered as a sustainedrelease composition or device, such as a Medipad™ device.

Agents of the invention can optionally be administered in combinationwith other agents that are effective in treating the disorder orcondition in need of treatment (e.g., prophylactic or therapeutic).Preferred additional agents are those which are art recognized and arestandardly administered for a particular disorder.

Effective single treatment dosages (i.e., therapeutically effectiveamounts) of 90Y-labeled antibodies of the invention range from betweenabout 5 and about 75 mCi, more preferably between about 10 and about 40mCi. Effective single treatment non-marrow ablative dosages of131I-labeled antibodies range from between about 5 and about 70 mCi,more preferably between about 5 and about 40 mCi. Effective singletreatment ablative dosages (i.e., may require autologous bone marrowtransplantation) of 131I-labeled antibodies range from between about 30and about 600 mCi, more preferably between about 50 and less than about500 mCi. In conjunction with a chimeric modified antibody, owing to thelonger circulating half life vis-a-vis murine antibodies, an effectivesingle treatment non-marrow ablative dosages of iodine-131 labeledchimeric antibodies range from between about 5 and about 40 mCi, morepreferably less than about 30 mCi. Imaging criteria for, e.g., the 111Inlabel, are typically less than about 5 mCi.

While a great deal of clinical experience has been gained with 131I and0.90Y, other radiolabels are known in the art and have been used forsimilar purposes. Still other radioisotopes are used for imaging. Forexample, additional radioisotopes which are compatible with the scope ofthe instant invention include, but are not limited to, 123I, 125I, 32P,57Co, 64Cu, 67Cu, 77Br, 81Rb, 81Kr, 87Sr, 113In, 127Cs, 129Cs, 132I,197Hg, 203Pb, 206Bi, 177Lu, 186Re, 212Pb, 212Bi, 47Sc, 105Rh, 109Pd,153Sm, 188Re, 199Au, 225Ac, 211A 213Bi. In this respect alpha, gamma andbeta emitters are all compatible with in the instant invention. Further,in view of the instant disclosure it is submitted that one skilled inthe art could readily determine which radionuclides are compatible witha selected course of treatment without undue experimentation. To thisend, additional radionuclides which have already been used in clinicaldiagnosis include 125I, 123I, 99Tc, 43K, 52Fe, 67Ga, 68Ga, as well as111In. Antibodies have also been labeled with a variety of radionuclidesfor potential use in targeted immunotherapy (Peirersz et al. Immunol.Cell Biol. 65: 111-125 (1987)). These radionuclides include 188Re and186Re as well as 199Au and 67Cu to a lesser extent. U.S. Pat. No.5,460,785 provides additional data regarding such radioisotopes and isincorporated herein by reference.

As previously discussed, the antibodies, or fragments thereof, of theinvention, can be administered in a pharmaceutically effective amountfor the in vivo treatment of mammalian disorders. In this regard, itwill be appreciated that the disclosed antibodies, or fragments thereof,will be formulated so as to facilitate administration and promotestability of the active agent. Preferably, pharmaceutical compositionsin accordance with the present invention comprise a pharmaceuticallyacceptable, non-toxic, sterile carrier such as physiological saline,non-toxic buffers, preservatives and the like. For the purposes of theinstant application, a pharmaceutically effective amount of a antibodyof the invention, conjugated or unconjugated to a therapeutic agent,shall be held to mean an amount sufficient to achieve effective bindingto a target and to achieve a benefit, e.g., to ameliorate symptoms of adisease or disorder or to detect a substance or a cell. In the case oftumor cells, the polypeptide will be preferably be capable ofinteracting with selected immunoreactive antigens on neoplastic orimmunoreactive cells and provide for an increase in the death of thosecells. Of course, the pharmaceutical compositions of the presentinvention may be administered in single or multiple doses to provide fora pharmaceutically effective amount of the polypeptide.

In keeping with the scope of the present disclosure, the antibodies ofthe invention may be administered to a human or other animal inaccordance with the aforementioned methods of treatment in an amountsufficient to produce a therapeutic or prophylactic effect. Thepolypeptides of the invention can be administered to such human or otheranimal in a conventional dosage form prepared by combining the antibodyof the invention with a conventional pharmaceutically acceptable carrieror diluent according to known techniques. It will be recognized by oneof skill in the art that the form and character of the pharmaceuticallyacceptable carrier or diluent is dictated by the amount of activeingredient with which it is to be combined, the route of administrationand other well-known variables. Those skilled in the art will furtherappreciate that a cocktail comprising one or more species ofpolypeptides according to the present invention may prove to beparticularly effective.

VI. Methods of Treating Kallidin or des-Arg10-Kallidin-AssociatedDisease or Disorders

The anti-Kallidin or des-Arg10-Kallidin antibodies, or fragmentsthereof, of the invention are useful for antagonizing Kallidin ordes-Arg10-Kallidin activity. Accordingly, in another aspect, theinvention provides methods for treating Kallidin ordes-Arg10-Kallidin-associated diseases or disorders by administering toa subject in need of thereof a pharmaceutical composition comprising oneor more anti-Kallidin or des-Arg10-Kallidin antibody, or antigen bindingfragment thereof of the invention.

Kallidin or des-Arg10-Kallidin-associated diseases or disorders amenableto treatment include, without limitation, pathophysiologic conditionssuch as inflammation, trauma, burns, shock, allergy, acute or chronicpain, and fibrosis, e.g., renal fibrosis. In certain exemplary,embodiments, antibodies of the invention may be issued to treat renalfibrosis and associated acute kidney injury as well as chronic kidneydiseases which are the main causes of end-stage renal failure.

One skilled in the art would be able, by routine experimentation, todetermine what an effective, non-toxic amount of antibody (or additionaltherapeutic agent) would be for the purpose of treating a Kallidin ordes-Arg10-Kallidin-associated disease or disorder. For example, atherapeutically active amount of a polypeptide may vary according tofactors such as the disease stage (e.g., stage I versus stage IV), age,sex, medical complications (e.g., immunosuppressed conditions ordiseases) and weight of the subject, and the ability of the antibody toelicit a desired response in the subject. The dosage regimen may beadjusted to provide the optimum therapeutic response. For example,several divided doses may be administered daily, or the dose may beproportionally reduced as indicated by the exigencies of the therapeuticsituation. Generally, however, an effective dosage is expected to be inthe range of about 0.05 to 100 milligrams per kilogram body weight perday and more preferably from about 0.5 to 10, milligrams per kilogrambody weight per day.

VII. Exemplification

The present invention is further illustrated by the following exampleswhich should not be construed as further limiting. The contents ofSequence Listing, figures and all references, patents and publishedpatent applications cited throughout this application are expresslyincorporated herein by reference.

Furthermore, in accordance with the present invention there may beemployed conventional molecular biology, microbiology, and recombinantDNA techniques within the skill of the art. Such techniques areexplained fully in the literature. See, e.g., Sambrook, Fritsch &Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989)Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds.(1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins,eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)];Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, APractical Guide To Molecular Cloning (1984); F. M. Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994).

EXAMPLE 1 Hybridoma Production: Immunization of Mice with KallidinPeptide Conjugated to KLH and Antibody Generation against Human BKR1Ligands

The objective was to develop cross-reactive antibodies against Kallidin(KD; SEQ ID NO:1) and des-arg-Kallidin (DAKD; SEQ ID NO:2) that wouldinhibit these ligands binding to the human BKR1. Generally, immunizationof mice with KLH conjugated KD through additional cysteines on eitherthe C- or N-terminus of the peptide was used to obtain mouse splenocytesfor fusion with mouse myeloma cell lines as a fusion partner to producethe hybridomas.

Briefly, the immunization protocol was as follows: BALB/c Mice (8-20week-old naïve female) were immunized intraperitoneally with a mixtureof even amounts of KLH-KD and KD-KLH in phosphate buffered saline (PBS)as an antigen total of 100 ug per mouse mixed at 1:1 ratio of SigmaAdjuvant System (Sigma cat #6322) in a total volume of 200 μl per mouse(day 0). On day 21, mice were boosted with a mixture of even amounts ofKLH-KD and KD-KLH in PBS as an antigen total of 50 ug per mouse mixed at1:1 ratio of Sigma Adjuvant System (Sigma cat #6322) in a total volumeof 200 μl per mouse. On day 30, blood samples were harvested for KDspecific antibody titer evaluation. On day 51, mice were boosted forfusion with a mixture of even amounts of KLH-KD and KD-KLH in PBS as anantigen total of 50 ug per mouse mixed at 1:1 ratio of Sigma AdjuvantSystem (Sigma cat #6322) in a total volume of 200 μl per mouse. At day55 mice were sacrificed by CO2 chamber, blood was collected through thecardiac puncture and spleen was harvested for hybridoma production.

Hybridomas were made by fusing mouse myeloma cells that are deficient inadenosine phosphoribosyltransferase (APRT) with spleen cells from miceimmunized with specific antigens. A selection system using HAT(hypoxanthine, azaserine, and thymidine) medium eliminates all but thefusion cells that are APRT+. Successful hybridomas must also retain theimmunoglobulin (Igh) heavy chain, one of the immunoglobulin light chainloci and secrete functional antibody.

Hybridoma Production Medium (IMDM) was made by combining the following:500 ml Iscove's Modified Dulbecco's Medium (HyClone SH30259.01), 50 mlfetal bovine serum (HyClone SH30070.03), 5 ml L-glutamine (GibcoInvitrogen cat #25030), 5 ml non-essential amino acids (Gibco Invitrogencat #11140050), 5 ml sodium pyruvate (Gibco Invitrogen cat #11360070), 5ml 0.1% penicillin-streptomycin (Gibco Invitrogen cat #15140148). Themedium was filtered before use. Expansion medium was made by combiningthe following: 1000 ml serum free medium (Gibco Hybridoma SFM #12045),100 ml 10% HyClone SuperLow IgG Defined FBS #SH30898.03 and 10 mlpenicillin/streptomycin. Freezing medium was 45 ml heat inactivated FBS(HyClone SH30070.03) and 5 ml DMSO, filter sterilized. Other materialsincluded the following: HAT (50×) was obtained from Sigma-Aldrich(#HO262); Hybridoma Fusion and Cloning Supplement (50×) (RocheDiagnostics 11 363 735 001); Trypan Blue Stain 0.4% (Invitrogen cat#15250-061 or T10282); PEG 1500 in 75 mM Hepes 50% w/v (Roche cat#783641(10783641001). All the reagents except HAT and Hybridoma Fusionand cloning supplement were used at 37° C.

TABLE 2 Peptide Reagents Used in Immunization and Screening PeptideSEQ ID Peptide Peptide Alternative No. NO. Sequence Name Name 1 5RPPGFSPFR bradykinin BK 2 117 biotin-RPPGFSPFR b-BK 3 71RPPGFSPFR-biotin BK-b 4 72 KLH-RPPGFSPFR KLH-BK 5 73 RPPGFSPFR-KLHBK-KLH 6 1 KRPPGFSPFR kallidin KD 7 74 biotin-KRPPGFSPFR b-KD 8 75KRPPGFSPFR-biotin KD-b 9 76 KLH-KRPPGFSPFR KLH-KD 10 77 KRPPGFSPFR-KLHKD-KLH 11 6 RPPGFSPF desArg9bradykinin DABK 12 78 biotin-RPPGFSPF b-DABK13 79 RPPGFSPF-biotin DABK-b 14 80 KLH-RPPGFSPF KLHDABK 15 81RPPGFSPF-KLH DABK-KLH 16 2 KRPPGFSPF desArg10kallidin DAKD 17 82biotin-KRPPGFSPF b-DAKD 18 83 KRPPGFSPF-biotin DAKD-b 19 84KLH-KRPPGFSPF KLH-DAKD 20 85 KRPPGFSPF-KLH DAKD-KLH 21 3 RRPPGFSPFRKallidin like peptide KLP 22 86 biotin-RRPPGFSPFR b-KLP 23 87RRPPGFSPFR-biotin KLP-b 24 88 KLH-RRPPGFSPFR KLH-KLP 25 89RRPPGFSPFR-KLH KLP-KLH 26 90 RRPPGFSPF desArg10kallidin like DAKLPpeptide 27 91 biotin-RRPPGFSPF b-DAKLP 28 92 RRPPGFSPF-biotin DAKLP-b 2993 KLH-RRPPGFSPF KLH-DAKLP 30 94 RRPPGFSPF-KLH DAKLP-b 31 95 RPPGFbradykinin1-5 BK15 32 96 biotin-RPPGF b-BK15

Briefly, three or four days before the fusion, the mouse was boostedwith an antigen of interest either intraperitonealy or intravenously. Onthe day of the fusion, the mouse was sacrificed in CO₂ chamber, bloodwas collected by cardiac puncture and the spleen was taken out andplaced into 10 ml of serum free IMDM in a Petri dish. Fusion partnercells myeloma: FO (ATCC ref CRL-1646)/X63 Ag8.653 (ATCC ref CRL1580)were grown at a log phase, then split one day before the fusion (1:2 and1:5), and collected into 20 ml centrifuge tubes, spun and resuspendedthe pellet in 10 ml IMDM. The pellet was washed two times with serumfree IMDM medium. All the centrifugations are performed at 1570 rpm for5 min. Final resuspension was in 10 ml serum free IMDM. The connectivetissue was dissected away from the spleen. The spleen was injected with1 ml of serum free IMDM preheated to 37° C. by 1 ml syringe and 25-gaugeneedle. Splenocytes are squeezed out of the fibroelastic coat by forcepsand washed two times in 10 ml of serum free IMDM (including initialspin) and were resuspended in 10 ml serum free IMDM. Cells were countedin Countess Automated Cell Counter.

Fusion partner cells and splenocytes were combined in one 50 ml tube atratio of 1:2 to 1:10 (by cell number) and spun down at 970 rpm for 10min (slow spin) to form a loose pellet. After the “slow” spin,supernatant was taken out with the precaution not to disturb the pellet,but minimize the amount of liquid over the cells in order not to dilutePEG 1500. The last remaining medium was reserved and added back afterthe PEG is added (below). Preheated PEG 1500 (37° C., total 1 ml) wasadded drop by drop to the cell pellet over 1 minute period of time andcells were mixed after every drop of PEG was added. Pellet was incubatedwith PEG for another 1 minute followed by addition of 10 ml ofserum-free IMDM medium over 1 minute, so that the first 1 ml out of 10is added over 30 sec. Cells underwent slow spin at 970 rpm for 10 minand supernatant decanted. Into (2) 100 ml troughs, the following wasadded: 70 ml IMDM with 10% FBS, 2 ml HATand 2 ml Hybridoma and FusionCloning Supplement. Cells were resuspended in 10 ml IMDM with 10% FBSand split into (2) 50 ml tubes (5 ml cells/tube) and 25 ml IMDM with 10%FBS was added. The resulting 30 ml was transferred to the troughscontaining 70 ml HBSS/HAT/cloning supplement and 200 ul cells/well werepipetted into (10) 96-well plates. Fusion was ready for screening byELISA (50 ul) about 10 to 14 days later, or when medium in the wellsturns yellow. After the primary screening, positive clones are selected,numbered and moved to a 24-well plate in 500 ul per well of IMDM with10% FBSH I. Hybridoma supernatants were screened by ELISA onstreptavidin plated coated with N- and C-term biotinylated peptides (seebelow).

EXAMPLE 2 Characterization and Selection of Hybridomas ExpressingAntibodies Against Human BKR1 Ligands

Hybridoma supernatants were screened by ELISA on streptavidin platedcoated with N- and C-term biotinylated peptides (see e.g., those setforth in Table 2) and then antibody binding kinetics were determined forconfirmed positive hybridoma clones.

The ability of the antibodies in hybridoma supernatants to bind to BKR1ligand peptide was evaluated with an ELISA assay. DAKD-biotin orKD-biotin peptides was coated on a 96-well SA plate in phosphatebuffered saline (PBS) buffer for an hour at room temperature at 5 ug/ml,and the nonspecific binding sites were blocked with 1% bovine serumalbumin (BSA) in PBS buffer. This plate was used to perform primary andsecondary screening of the crude hybridoma supernatants.

Hybridoma supernatants were added to the plates for binding to thecoated KD or DAKD peptides. After 1 hour incubation, the plate waswashed and bound antibodies were detected using horseradish peroxidase(HRP) conjugated secondary antibody (HRP-goat anti-mouse IgG (H+L):Jackson ImmunoResearch Labs #115-035-166) and developed using2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) substrate(Roche diagnostics #11 204 521 001). Data was analyzed using Excel. Theantibodies showing positive signals (2 fold higher than 1:10000 serumdilution ELISA signal) were selected and re-screened in duplicates forconfirmation. Confirmed positive hybridoma clones were selected andsubjected to binding dissociation rate ranking by Biacore.

For antibody binding kinetics, the instruments used were the BIACORE2000 or BIACORE 3000 (GE Healthcare), designed for biomolecularinteraction analysis (BIA) in real time. The sensor chip used was SAchip (GE Healthcare) with streptavidin covalently immobilized on acarboxymethylated dextran matrix. Each sensor chip has four parallelflow cells (Fc). Every biotinylated BKR1 or BKR2 ligand peptides wereimmobilized to one of the flow cells 2 to 4 (Fc2 to Fc4) in the SA chipfor binding dissociation rate screening and selectivity screening. Flowcell 1 (Fc1) was reserved and immobilized with a random peptide(biotinylated at one terminus) with equal or close peptide length incomparison to the testing ligand peptides as the negative control. Inscreening assays, cell culture supernatants of the hybridoma clonesselected through primary screening or of transiently expressed humanizedvariants were injected over immobilized peptides. Hybridoma cell culturemedia was also injected over the chip surface as blank to establish abaseline. After subtracting signals of Fc1 and blank buffer runs, thedissociation rate of the antibodies from the supernatants to eachpeptide was analyzed and ranked using BIAevaluation software. Only theantibody clones that demonstrated superior (kd<10-4 1/s) bindingdissociation rate were selected for subcloning and furthercharacterization. In kinetics analysis, the correspondingbiotin-peptides identified in screening for the testing antibody wereimmobilized in Fc2 to Fc4 while Fc1 with a random peptide used asreference cell. Each purified antibody selected from screenings weremade into a series of two fold dilutions in running buffer (1× HBS-EPbuffer, GE Healthcare) between 0.1 to 10 nM. Binding association rate,dissociation rate and the overall affinity were calculated inBIAevaluation. Antibody binding kinetics for each antibody was alwaysconfirmed in triplicate assays using Biacore.

A total of 8 mice were immunized with mixed KLH-KD/KD-KLH andKLH-DAKD/DAKD-KLH and the spleens were fused using the above protocols.After primary screening of about 7680 hybridoma clones in ELISA withDAKD-biotin and KD-biotin, only 76 clones were confirmed positive andselected for binding dissociation rate ranking in Biacore 3000/2000 overthe immobilized DAKD-biotin and KD-biotin on Streptavidin (SA) chips.Among those, 8 hybridoma clones with binding dissociation rate <=of 10⁻⁴were subcloned, sequenced, purified and further characterized (see Table3).

TABLE 3 Immunization Results with KLH-KD/KD-KLH and KLH-DAKD/DAKD-KLHPeptide Used in Clone ID Ligand Assay Assay B21 C63 F151 F306 I2 I8I22** I54** DAKD b-DAKD ELISA + + − − + + + + Biacore − − − − − − NS NS(KD, M) DAKD-b ELISA + + + + + + + + Biacore 4.15E−11 1.42E−10 1.60E−101.60E−10 1.10E−10 6.25E−10 NTD NTD (KD, M) DAKD FLIPR 22-25, 9 − 6.9 6.99.4 8.1 (50 nM) (nM) DABK b-DABK ELISA +/− +/− − − + + + + Biacore − − −− − − NS NS (KD, M) DABK-b ELISA + +/− − − + + + + Biacore −* − − − − −NS NS (KD, M) DABK FLIPR (nM) BK b- ELISA − − − − − − − − BK Biacore − −− − − − NS NS (KD, M) BK-b ELISA + − − − + + + + Biacore 8.57E−09 − − −− − NS NS (KD, M) BK FLIPR − − − − (nM) KD b-KD ELISA + − − − − − − −Biacore − − − − − − NS NS (KD, M) KD-b ELISA + + + + + + + + Biacore 8.4E−11 2.21E−10  2.9E−11  2.9E−11  7.8E−11 7.53E−10 NTD NTD (KD, M) KDFLIPR 12 − 3.0 3.0 7.3 8.3 25   30   (15 nM)M) (nM) NA = not applicable,negative in ELISA NS = nonspecific binding NTD = not to be determined −*= residual binding (low RU in Biacore)

Based on results seen in Table 3, five clones with unique sequences wereselected for kinetic studies. These antibodies were highly selective forDAKD-biotin, KD-biotin, DAKLP-biotin and KLP-biotin binding (see Table4). They do not bind to other kinin peptides or to peptides biotinylatedat the N-terminus.

TABLE 4 Summary of Kinetics of Selected anti-DAKD/KD Antibody CandidatesDAKD-b KD-b Antibody koff KD koff KD C63 9.36E−05 1.42E−10 1.00E−042.21E−10 B21 9.89E−05 4.15E−11 2.04E−04 8.40E−11 F151 1.36E−04 1.62E−102.00E−05 2.88E−11 I22 3.19E−04 2.17E−10 2.10E−05 4.40E−12 I54 3.06E−059.53E−12 3.88E−05 1.12E−11 DAKLP-b KLP-b koff KD koff KD C63 n/b n/b n/bn/b B21 2.30E−04 1.34E−10 1.12E−04 1.92E−10 F151 6.58E−05 2.12E−10≦1.0E−06  ≈1.66E−11 I22 ≦1.0E−06  ≈1.83E−12 1.03E−05 1.82E−12 I545.66E−05 1.17E−11 6.04E−05 9.56E−12 n/b = no binding

Additional immunization were performed with an array of immunogens (seelist of peptides, Table 2) for generating antibodies blocking the rodentBKR1 ligands, DABK and DAKD as well as antibodies with other bindingspecificities against different member of kinin family of peptides.Table 5 lists the heavy and light sequences of the antibodies generated.

TABLE 5 Heavy and Light Chain Sequences of Antibodies Antibody IsotypeSEQ ID NO. Heavy Chain Sequence B21 IgG1/k  97LPEFQVKLEESGAELVRSGASVKLSCTAS GFNIKDYYLH WVKQRPEQGLEWIG WIDPENGDTGYARKFQG KATMTADTSSNTVYLHLSSLTSEDTAVYYFNA WEYDGYYD LDYWGQGTSVTVSSAKTTPPSVYGSS C63  98 LPEFQVQLEESGGGLVQPGGSMKLSCVAS GFTFSNYWMNWVRQSPEKGLEWVA E IRSKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCIGEDYGGDY WGQGTSVTVSSAKTTPPSVYGSS F151  99 LPEFEVQLEESGPELVKPGTSVKVSCKASGYSFTDYNIY WVKQSHGKSLEWIG YF DPYNGNTGYNQKFRGKATLTVDKSSSTAFMHLSSLTSDDSAVYYCAN YYRYDDHA MDY WGQGTSVTVSSAKTTPPSVYGSSI22 100 LPEFEVKLQESGAELVRSGASVKLSCTAS GFNIKDYYMH WVKQRPEQGLEWIG WVDPENGDSDYAPKFQG KATMTADTSSNTVYLQFSSLTSEDTAVYYCNA FEYDGNYS SLDFWGQGTSVTVSSAKTTPPSVYGSS 154 101 LPEFEVKLEQSGAELVRSGASVKLSCTAS GFNIKDYYMHWVKQRPEQGLEWIG WV DPENGDSDYAPKFQG KATMTADTSSNTVYLQFSSLTSEDTAVYYCNAFEYDGNYS PLDF WGQGTSVTVSSAKTTPPSVYGSS B21 mIgG1/K 118EVQLQQSGAELVRSGASVKLSCTASGFNIKDYYLH WVKQRPEQGLEWIG WIDPEN GDTGYARKFQGKATMTADTSSNTVYLHLSSLTSEDTAVYYFNAWEYDGYYDLDY W GQGTSVTVSSAKTTPPS C63 119EVKLEESGGGLVQPGGSMKLSCVASGFTFSNYWMN WVRQSPEKGLEWVA EIRSKS NNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCIGEDYGGDY WGQ GTSVTVSSAKTTPPS F151 120EIQLQQSGPELVKPGTSVKVSCKASGYSFTDYNIY WVKQSHGKSLEWIG YFDPYN GNTGYNQKFRGKATLTVDKSSSTAFMHLSSLTSDDSAVYYCANYYRYDDHAMDY W GQGTSVTVSSAKTTPPS I22 121EVQLQQSGAELVRSGASVKLSCTASGFNIKDYYMH WVKQRPEQGLEWIG WVDPEN GDSDYAPKFQGKATMTADTSSNTVYLQFSSLTSEDTAVYYCNAFEYDGNYSPLDF WGQGTSVTVSSAKTTPPS 154 122EVQLQQSGAELVRSGASVKLSCTASGFNIKDYYMHWVKQRPEQGLEWIGWVDPEN GDSDYAPKFQGKATMTADTSSNTVYLQFSSLTSEDTAVYYCNAFEYDGNYSPLDF WGQGTSVTVSSAKTTPPS AntibodyIsotype SEQ ID NO. LightChainSequence B21 IgG1/k 102ELDIVMTQTTLTLSVTIGQPASISC KSSQSLLYSNGKTYLN WLLQRPGQSPKRLI Y LVSKLDSGVPDRFTGSGSGTDFTLKIIRVEAEDLGVYYC LQGTHEPYT FGGGTKLEIKRADAAPTVSIFPPSKLELY C63 103 ELDIVLTQSPSSLAVSVGEKVTMSCKSSQSLLYSSDQRNYLA WYQQRSGQSPKLL IY WASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYC QQYYSYPYT FGGGTKLEIKRADAAPTVSIFPPSKLELY F151 104ELDIVMTQTPSSLAVSVGEKVTMSCKSSQSLLYTSNQKNYLAWYQQKPGQSPKPL IY WASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAIYYC QQYYSYPWT EGGGTKLEIKRADAAPTVSIFPPSKLELY I22 105 ELDIVITQTTLSLSVPIGQPASISCKSRQSLLYSNGETYLN WLLQRPGQSPKRLI Y LVSKLDSGVPDRFTGSRSGTDFTLKISRVESEDLGVYYC MQGTHEPYT FGGGTKLEIKRADAAPTVSIFPPSKLELY 154 106 ELDIVITQSTLTLSVPIGQPASISCKSSQSLLYSNGETYLN WLL QRPGQSPKRQI YLVSKLDSGVPDRFTGSRSGTDFTLKISRVESEDLGVYYCMQGTHEPYTFGGGTKLEIKRADAAPTVSIFPPSKLELY B21 mIgG1/K 123 DVVMTQTPLTLSVTIGQPASISCKSSQSLLYSNGKTYLN WLLQRPGQSPKRLIY L VSKLDSGVPDRFTGSGSGTDFTLKIIRVEAEDLGVYYC LQGTHEPYT F GGGTKLE IKRADAAPT C63 124DIVMSQSPSSLAVSVGEKVTMSC KSSQSLLYSSDQRNYLA WYQQRSGQSPKLLIY WASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYC QQYYSYPYT FGGGTKL EIKRADAAPT F151 125DIVMSQSPSSLAVSVGEKVTMSC KSSQSLLYSSNQKNYLA WYQQKPGQSPKPLIY WASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAIYYC QQYYSYPWT FGGGTKL EIKRADAAPT I22 126DIVMTQSPSSLSVSAGEKVTMSC KSSQSLLNSGNQKNYLA WYQQKPGQPPKLLIY GASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC QNDHSYPLT FGAGTKL ELKRADAAPT 154 131DVVMTQTPLTLSVPIGQPASISCKSSQSLLYSNGETYLNWLLQRPGQSPKRLIYL VSKLDSGVPDRFTGSRSGTDFTLKISRVESEDLGVYYC MQGTHEPYT FGGGTKLE IKRADAAPT Singleunderscore = CDR region; double underscore = signature amino acids foridentifying CDRs

EXAMPLE 3 Generation of Surrogate Antibody for Murine Animal Studies

A surrogate antibody to be used in murine animal studies needed to beable to bind and neutralize rodent BKR1 ligands, DABK and DAKLP (mouseequivalent of DAKD). In order to generate the required surrogateantibody, mice were first immunized with DABK and/or DAKD with KLHdirectly conjugated to the N-terminals of the peptides.Biotin-DABK/biotin-DAKD (biotinylation directly on N-terminus of thepeptide) positive hybridoma clones from ELISA screening were selectedfor scaling up and purification. The antibodies listed in Family 7 (seeTable 12) that demonstrated high binding affinities to biotin-DABK,biotin-DAKLP and biotin-DAKD were selected based on Biacore directbinding assay (Table 10). However, these Family 7 antibodies showed nobinding to the native, unmodified DABK and DAKD peptides in competitiveELISA, and lacked neutralizing functionality in a calcium influx assaywith Functional Drug Screening System (FDSS) (Hamamatsu Photonics K.K.,Japan). Moreover, the biotin-DABK and biotin-DAKD completely lostbioactivity in the FDSS assay in comparison to the native, unmodifiedDABK and DAKD peptides (data not shown).

It was hypothesized that the direct N-terminus conjugation of KLH andbiotin prevented the native confirmation of DABK and DAKD to form. Withthe aim to restore the native conformation in KLH- and biotin-conjugatedpeptides, linkers were designed and added to the N-terminus of DABKand/or DAKD with the intention to “cushion” the KLH/biotin conjugationeffects on peptide conformation. Poly-glycine linkers were firstattempted and tested because of their simple, non-polar and neutralproperties based on modeling results. The FDSS assay results indicatedthat the gly-gly-gly (3G) linker was the best according to its abilityto restore the bioactivities of KLH and biotin conjugated DABK and DAKDpeptides (data not shown). Therefore, KLH-3G-DABK was chosen to immunizemice. And biotin-3G-DABK and biotin-3G-KD were used in binding basedscreening assays (ELISA and Biacore). Several DABK/DAKD specificantibodies (Family 3, see Table 13) were identified in this new round ofsurrogate antibody hybridoma selection. EE1 was selected as the leadsurrogate antibody based on its superior binding affinity andneutralization activity against native DABK/DAKD and lack ofcross-reactivity to other peptides (see Tables 6-12)

Antibodies with different specificities were generated when using thedifferent immunogens listed in Table 13. Family 4 antibodies werespecific to the BKR2 receptor ligands, BK and KD. Family 5 antibodiesspecifically bind to the C terminus of BK and DABK. Family 6 antibodiesbind BK, DABK and DAKD but do not bind to KD.

Additional linkers were evaluated for binding to the surrogate EE1antibody for their ability to fit into the DABK/DAKD binding pocket inEE1, including longer poly-glycine linkers, poly-alanine linkers andpreexisting linkers such as polyethylene glycol (PEG2) linker andaminohexanoic acid (Ahx) linker (a 6-carbon inert linker). All linkerpeptides were custom synthesized by Abgent (Can Diego, Calif.). Alltested biotinylated peptides with linkers (biotin-linker-DABK/DAKD)bound well to EE1, indicating that any inert N-terminus linkers helpedDABK and DAKD peptides to retain their native bioactive conformationwhen conjugated with biotin and other molecules. In contrast, no bindingor poor binding to EE1 was observed with biotin-DABK and biotin-DAKD,peptides that have direct N-terminal biotin conjugation (see FIG. 1).

The binding kinetics of generated antibodies are summarized in Tables5-11. Then, all antibodies generated were sorted into families and theirbinding specificities are summarized below in Table 12. Table 13provides the heavy and light chain sequences of antibodies that wereplaced into family 1 and family 2 based on their binding specificity(see Table 12).

TABLE 6 Summary of Antibody Kinetics to b-3G-DABK and b-3G-DAKD Peptidesb-3G-DABK b-3G-DAKD Clone Ka (1/Ms) Kd (1/s) KD (M) Ka (1/Ms) Kd (1/s)KD (M) DD20 1.5E+06 2.3E−04 1.6E−10 4.7E+05 4.0E−04 8.8E−10 UR11 2.0E+053.0E−04 1.5E−09 3.0E+05 1.6E−03 5.2E−09 DD7 2.3E+05 6.0E−04 2.7E−092.1E+05 1.4E−03 6.6E−09 EE1 4.4E+05 1.2E−04 2.8E−10 4.4E+05 2.0E−044.5E−10 EE36 4.3E+03 5.3E−04 1.2E−07 n/b n/b n/b UR29 n/b* n/b n/b n/bn/b n/b JK3 3.44E+05  3.91E−05  1.14E−10  3.18E+05  5.07E−05  1.60E−10 LR4 n/b n/b n/b n/b n/b n/b LR16 n/b n/b n/b n/b n/b n/b LR6 n/b n/b n/bn/b n/b n/b LR12 n/b n/b n/b n/b n/b n/b NR1 n/b n/b n/b n/b n/b n/bNR15 n/b n/b n/b n/b n/b n/b n/b* = non-specific binding, n/b = nobinding

TABLE 7 Summary of Antibody Kinetics to b-3G-DAKLP and b-3G-BK Peptidesb-3G-DAKLP b-3G-BK Clone Ka (1/Ms) Kd (1/s) KD (M) Ka (1/Ms) Kd (1/s) KD(M) DD20 3.1E+05 6.1E−04 2.0E−09 3.0E+05 7.1E−04 2.3E−09 UR11 n/b n/bn/b n/b n/b n/b DD7 1.7E+05 2.1E−03 1.2E−08 1.3E+05 8.8E−04 6.8E−09 EE14.2E+05 2.9E−04 6.8E−10 2.5E+05 2.6E−03 1.1E−08 EE36 n/b n/b n/b n/b n/bn/b UR29 n/b n/b n/b n/b n/b n/b JK3 ND ND ND n/b n/b n/b LR4 n/b n/bn/b n/b n/b n/b LR16 n/b n/b n/b n/b n/b n/b LR6 n/b n/b n/b n/b n/b n/bLR12 n/b n/b n/b n/b n/b n/b NR1 n/b n/b n/b n/b n/b n/b NR15 n/b n/bn/b n/b n/b n/b n/b = no binding; ND = not determined

TABLE 8 Summary of Antibody Kinetics to b-3G-KLP and b-3G-KD Peptidesb-3G-KLP b-3G-KD Clone Ka(1/Ms) Kd(1/s) KD(M) Ka(1/Ms) Kd(1/s) KD(M)DD20 3.8E+05 5.7E−04 1.5E−09 n/b n/b n/b UR11 n/b n/b n/b 1.2E+051.3E−03 1.1E−08 DD7 1.5E+05 2.1E−03 1.5E−08 2.2E+05 1.8E−03 8.4E−09 EE14.0E+05 2.1E−03 5.3E−09 n/b n/b n/b EE36 n/b n/b n/b n/b n/b n/b UR29n/b n/b n/b n/b n/b n/b JK3 ND ND ND n/b n/b n/b LR4 n/b n/b n/b n/b n/bn/b LR16 n/b n/b n/b n/b n/b n/b LR6 n/b n/b n/b n/b n/b n/b LR12 n/bn/b n/b n/b n/b n/b NR1 n/b n/b n/b n/b n/b n/b NR15 n/b n/b n/b n/b n/bn/b n/b = no binding; ND = not determined

TABLE 9 Summary of Antibody Kinetics to DABK-b and DAKLP-b PeptidesDABK-b DAKLP-b Clone Ka(1/Ms) Kd(1/s) KD(M) Ka(1/Ms) Kd(1/s) KD(M) DD20n/b n/b n/b n/b n/b n/b UR11 n/b n/b n/b n/b n/b n/b DD7 n/b n/b n/b n/bn/b n/b EE1 n/b n/b n/b n/b n/b n/b EE36 n/b n/b n/b n/b n/b n/b UR291.5E+06 5.8E−05 3.9E−11 3.0E+06 2.1E−03 6.8E−10 JK3 n/b n/b n/b n/b n/bn/b LR4 n/b n/b n/b n/b n/b n/b LR16 n/b n/b n/b n/b n/b n/b LR6 n/b n/bn/b n/b n/b n/b LR12 n/b n/b n/b n/b n/b n/b NR1 n/b n/b n/b n/b n/b n/bNR15 n/b n/b n/b n/b n/b n/b n/b = no binding; ND = not determined

TABLE 10 Summary of Antibody Kinetics to BK-b and b-BK Peptides BK-bb-BK Clone Ka(1/Ms) Kd(1/s) KD(M) Ka(1/Ms) Kd(1/s) KD(M) DD20 n/b n/bn/b n/b n/b n/b UR11 n/b n/b n/b n/b n/b n/b DD7 n/b n/b n/b n/b n/b n/bEE1 n/b n/b n/b n/b n/b n/b EE36 n/b n/b n/b n/b n/b n/b UR29 1.5E+061.0E−04 7.2E−11 n/b n/b n/b JK3 n/b n/b n/b n/b n/b n/b LR4 n/b n/b n/bn/b n/b n/b LR16 n/b n/b n/b n/b n/b n/b LR6 n/b n/b n/b n/b n/b n/bLR12 n/b n/b n/b n/b n/b n/b NR1 n/b n/b n/b 8.69E+04 8.77E−04 1.01E−08NR15 n/b n/b n/b 2.95E+05 1.09E−03 3.68E−09 n/b = no binding; ND = notdetermined

TABLE 11 Summary of Antibody Kinetics to b-DABK and b-DAKD Peptidesb-DABK b-DAKD Clone Ka(1/Ms) Kd(1/s) KD(M) Ka(1/Ms) Kd(1/s) KD(M) DD20n/b n/b n/b n/b n/b n/b UR11 n/b n/b n/b n/b n/b n/b DD7 n/b n/b n/b n/bn/b n/b EE1 n/b n/b n/b n/b n/b n/b EE36 n/b n/b n/b n/b n/b n/b UR29n/b n/b n/b n/b n/b n/b JK3 n/b n/b n/b n/b n/b n/b LR4 1.48E+051.04E−03 7.15E−09 3.27E+05 7.63E−04 2.36E−09 LR16 4.34E+05 4.38E−051.01E−10 2.07E+05 3.39E−03 1.65E−08 LR6 n/b n/b n/b n/b n/b n/b LR122.91E+05 5.40E−04 3.63E−09 n/b n/b n/b NR1 n/b n/b n/b n/b n/b n/b NR15n/b n/b n/b n/b n/b n/b n/b = no binding; ND = not determined

TABLE 12 Summary of Antibody Kinetics to b-DAKLP and b-KD Peptidesb-DAKLP b-KD Clone Ka(1/Ms) Kd(1/s) KD(M) Ka(1/Ms) Kd(1/s) KD(M) DD20n/b n/b n/b n/b n/b n/b UR11 n/b n/b n/b n/b n/b n/b DD7 n/b n/b n/b n/bn/b n/b EE1 n/b n/b n/b n/b n/b n/b EE36 n/b n/b n/b n/b n/b n/b UR29n/b n/b n/b n/b n/b n/b JK3 n/b n/b n/b n/b n/b n/b LR4 1.84E+052.58E−04 1.40E−09 n/b n/b n/b LR16 2.34E+05 1.11E−04 4.74E−10 n/b n/bn/b LR6 6.80E+05 4.01E−04 7.45E−10 n/b n/b n/b LR12 n/b n/b n/b n/b n/bn/b NR1 n/b n/b n/b 1.66E+05 5.81E−03 3.56E−08 NR15 n/b n/b n/b 7.66E+055.66E−03 7.41E−09 n/b = no binding; ND = not determined

TABLE 13 Summary of Anti-kinin peptide antibody generation AntibodyRepresentative Families Immunogens antibodies Binding Specificity Family1 KD-KLH + F151, B21, I22, N-terminus of DAKD KLH-KD or I54 (DAKLP) andKD DAKD-KLH + (KLP) KLH-DAKD Family 2 KD-KLH + C63 N-terminus of DAKDKLH-KD or and KD DAKD-KLH + KLH-DAKD Family 3 KLH-3G- EE1, DD20, JK3C-terminus of DABK DABK and DAKD (DAKLP) Family 4 KLH-BK and NR15, NR1C-terminus of BK and BSA-BK KD Family 5 KLH-BK UR29 N-terminus of BK andDABK Family 6 KLH-BK UR11 BK, DABK and DAKD Family 7 KLH-DABK LR4, LR6,LR12 no binding with native or KLH-DAKD and LR16 peptides

EXAMPLE 4 Characterization of des-arg-Kinin Ligand Depletion usingCalcium Mobilization

A functional assay was used to further characterize the seven familiesof generated antibodies. The Bradykinin B1 Receptor signaling is Gqcoupled, therefore receptor activation can be monitored using Gqactivation of IP3 and downstream calcium mobilization. HEK mBKR1(recombinant mouse bradykinin B1 receptor) cells or MRC5 (endogenousexpression of bradykinin B2 receptors; (ATCC CCL-171)) were used tomeasure calcium mobilization.

Briefly, the mouse Bdkrb1 gene (sequence provided below) was amplifiedfrom mouse lung cDNA (Biochain, Cat #C1334152) using PCR primers804_cGWY_F:

(SEQ ID NO: 107) 5′-AAAAGCAGGCTTAGGAGCGGCCGCCATGGCGTCCCAGGCCTCGCTG-  3′

and 804_cGWY_R:

(SEQ ID NO: 108) 5′-CAAGAAAGCTGGGTCGGATCCTTATAAAGTTCCCAG AACCCTGGTC-3′

and Pfu Polymerase (Agilent Technologies, Cat #600264) and cloned intopDONR201 using BP clonase enzyme mix (Invitrogen, Cat #11789-020). Inparallel, the pEAK8 expression vector (EDGE Biosystems) was modified byinserting a N-terminal HA tag (GCATACCCATACGACGTCCCAGACTACGCT, GenBankSEQ ID NO:109 CY100443) into pEAK8 linearized with EcoRI and Hind III(vector pEAK8-nHA) and subsequent insertion of the Gateway cassette B(Invitrogen, Cat #11828-029) into pEAK8_nHA digested with EcoRI and NotIand blunt-ended with Klenow polymerase (NEB, cat #M0210S) resulting invector pEAK8_nHA_DEST. Next mouse Bdkrb1 was subcloned intopEAK8_nHA_DEST using LR clonase (Invitrogen, Cat #11791-100). 293-PSCcells were then transfected with pEAK8-Bdkrb1 plasmid using Fugene 6transfection reagent. The cells were put under antibiotic (puromycin)selection 24 hours after transfection, and selection was maintained togenerate a stable cell line. Presence of the Bdkrb1 gene in theresultant stable cell lines was confirmed using real time RT-PCR, and byagarose gel electrophoresis. Cell surface expression of the BradykininB1 receptor was performed by using an antibody against the N-terminal-HAtag (Covance, Cat #MMS-101P) on the Bradykinin B1R on a FACS instrument.Functional activity of the Bradykinin B1 receptor was demonstrated incalcium mobilization assay with selective agonists.

Bdkrb1 gene subcloned into cells:

(GenBank NM_007539; SEQ ID NO: 110)ATGGCGTCCCAGGCCTCGCTGAAGCTACAGCCTTCTAACCAAAGCCAGCAGGCCCCTCCCAACATCACCTCCTGCGAGGGCGCCCCGGAAGCCTGGGATCTGCTGTGTCGGGTGCTGCCAGGGTTTGTCATCACTGTCTGTTTCTTTGGCCTCCTGGGGAACCTTTTAGTCCTGTCCTTCTTCCTTTTGCCTTGGCGACGATGGTGGCAGCAGCGGCGGCAGCGCCTAACCATAGCAGAAATCTACCTGGCTAACTTGGCAGCTTCTGATCTGGTGTTTGTGCTGGGCCTGCCCTTCTGGGCAGAGAACGTTGGGAACCGTTTCAACTGGCCCTTTGGAAGTGACCTCTGCCGGGTGGTCAGCGGGGTCATCAAGGCCAACCTGTTCATCAGCATCTTCCTGGTGGTGGCCATCAGTCAGGACCGCTACAGGTTGCTGGTATACCCCATGACCAGCTGGGGGAACCGGCGGCGACGGCAAGCCCAAGTGACCTGCCTGCTCATCTGGGTAGCTGGGGGCCTCTTGAGCACCCCCACGTTCCTTCTGCGTTCCGTCAAAGTCGTCCCTGATCTGAACATCTCTGCCTGCATCCTGCTTTTCCCCCACGAAGCTTGGCACTTTGTAAGGATGGTGGAGTTGAACGTTTTGGGTTTCCTCCTCCCATTGGCTGCCATCCTCTACTTCAACTTTCACATCCTGGCCTCCCTGAGAGGACAGAAGGAGGCCAGCAGAACCCGGTGTGGGGGACCCAAGGACAGCAAGACAATGGGGCTGATCCTCACACTGGTAGCCTCCTTCCTGGTCTGCTGGGCCCCTTACCACTTCTTTGCCTTCCTGGATTTCCTGGTCCAGGTGAGAGTGATCCAGGACTGCTTCTGGAAGGAGCTCACAGACCTGGGCCTGCAGCTGGCCAACTTCTTTGCTTTTGTCAACAGCTGCCTGAACCCACTGATTTATGTCTTTGCAGGCCGGCTCTTTAAGACCAGGGTTCTGGGAACTTTATAA

HEK mBKR1 or MRC5 cells were plated into 384 well clear bottom plates ingrowth medium, and allowed to attach overnight. Then growth media wasremoved, cells were washed in assay buffer (HBSS, 20 mM HEPES, 2.5 mMprobenecid), then dye-loaded with 0.5 uM Fluo-4AM, a cell permeablecalcium sensing dye, with 0.04% Pluronic Acid for 1 hr at 37 C. The AMester is cleaved, and the calcium dye is retained in the cytoplasm.After 1 hr, the cells were washed to remove excess dye, and 20 ul ofresidual buffer remained on the cells. Treatments were added as 2×solutions on the Functional Drug Screening System from Hamamatsu (FDSS),and the calcium mobilization was monitored kinetically for at least 4minutes. B1 R or B2R receptor activation results in Galpha q mediatedactivation of phospholipase C and IP3 mediated calcium mobilization. TheFluo-4 dye chelates the released calcium, and a robust change influorescence is observed. The results were exported as max-min relativefluorescence units to normalize for differences between cell density ordye loading across the plate.

Ligand potency was determined each day by running concentration responsecurves of ligand, and an approximate EC70-80 concentration of ligand wasselected for incubation with antibodies. An EC80 concentration wasselected because it is on the linear range of the detection curve andthere was ample window to see a decrease with antagonists or liganddepleting antibodies. Dose response curve of antibodies were allowed tobind a EC80 concentration ligand, and the extent of ligand depletion wasmonitored using change in fluorescence. Results were normalized tobuffer and EC80 ligand response, and an EC50 for ligand depletion wascalculated. The results were then reported as molar ratio whichcorresponds to the Antibody concentration that reduces depletes 50% ofthe ligand response (i.e., EC50 of Ab) divided by the ligandconcentration used. The theoretical max should be 0.5 because one unitof antibody should be able to deplete 2 units of ligand, but we haveseen lower values in practice but that may be a reflection of theinsensitivity of the detection method for low ligand concentrations,rather than a stochiometric constraint for the antibody. The results ofthese experiments are set forth in Tables 14-16.

All family 1 and family 2 antibodies (see Table 13) demonstratedsuperior binding kinetics by Biacore (Table 3) and neutralizationactivity as measured by calcium mobilization against DAKD and KDpeptides (Tables 14 and 15). The antibodies were further analyzed fortheir thermal stability and sequence suitability for humanization. F151was advanced for humanization because it was thermally stable, therewere no problematic residues in the CDR regions and it wascross-reactive to the mouse ligand KLP and DAKLP.

TABLE 14 Characterization of des-arg-Kinin Ligand Depletion usingCalcium Mobilization in HEK mBKR1 cells Depletion of DABK Depletion ofDAKD Depletion of DAKLP DABK DAKD DAKLP (Mean SD (Mean SD (Mean SD MolarMolar Molar Molar Molar Molar Family Antibody Ratio) Ratio n Ratio)Ratio n Ratio) Ratio n 1 F151 IA100 5 0.08 0.04 7 0.15 0.04 4 1 B21IA100 1 0.15 0.04 3 0.67 1 1 I22 IA100 1 0.07 0.02 3 0.21 1 1 I54 IA1001 0.15 0.05 3 0.35 1 2 C63 IA100 1 0.08 0.02 3 5.85 1 3 EE1 1.03 0.52 50.86 0.52 3 0.57 0.36 4 3 DD20 3.45 1.34 3 1.82 0.76 3 1.31 0.86 3 DD72.18 0.45 3 4.22 0.95 3 5.34 1.22 2 3 JK3 1.86 0.03 2 ND 1.44 0.03 2 4MBK3 ND ND ND 4 NR15 ND ND ND 4 NR1 ND ND ND 5 UR29 0.60 0.12 5 IA200 3IA300 4 6 UR11 6.99 1.61 3 19.65  14.95 3 11.09  3.13 2 7 LR4 IA100 1IA400 1 IA400 1 7 LR6 IA100 1 IA100 1 ND 7 LR12 IA100 1 IA100 1 ND 7LR16 IA100 1 IA100 1 ND Antibodies were pre-incubated with a setconcentration of ligand, usually an EC70-80 for activating calciummobilization at the Bradykinin B1 Receptor. The antibody-ligand mixturewas added to HEK mBKR1 cells pre-loaded with a calcium sensing dye(Fluo-4AM or Fluo-8AM) on the Hamamatsu FDSS6000 instrument, and calciummobilization was monitored. Data was exported as a max-min relativefluorescence of the biological response, and IC50 for ligand depletionwas calculated using sigmoidal curve fit in Graph Pad Prism V4.03. Datareported as molar ratio for ligand depletion by the antibody tostandardize the different concentration of ligand that was used for thevarious experiments. Molar Ratio for ligand depletion = [IC50 ofAntibody]/[Ligand] SD = Standard Deviation; ND = not determined; IA100 =Inactive at 100 nM; IA200 = Inactive at 200 nM; IA300 = Inactive at 300nM; IA400 = Inactive at 400 nM

TABLE 15 Characterization of Kinin Ligand Depletion using CalciumMobilization in MRC5 Fetal Lung Fibroblasts cells Depletion of BKDepletion of KD Depletion of KLP BK KD KLP (Mean SD (Mean SD (Mean SDMolar Molar Molar Molar Molar Molar Family Antibody Ratio) Ratio nRatio) Ratio n Ratio) Ratio n 1 F151 IA100 5 0.14 0.05 5 0.15 0.02 3 1B21 IA100 1 0.33 1 ND 1 I22 IA100 1 0.22 1 ND 1 I54 IA100 1 0.30 1 ND 2C63 IA100 1 0.23 1 ND 3 EE1 IA300 4 IA300 4 IA150 1 3 DD20 IA600 4 IA6005 IA150 1 DD7 7.11 3.62 3 17.37  12.11 3 4.27 1 3 JK3 IA300 2 IA300 2 ND4 MBK3 22.11  14.10 9 3.46 2.64 6 9.45 1 4 NR15 15.26  11.51 5 4.34 2.555 11.18  1 4 NR1 39.31  1 42.15  1 32.58  1 5 UR29 1.15 0.86 5 0.30 0.082 0.41 1 6 UR11 5.41 0.80 2 25.21  4.54 2 1.53 1 7 LR4 IA100 1 IA100 1ND 7 LR6 IA100 1 IA100 1 ND 7 LR12 IA100 1 IA100 1 ND 7 LR16 IA100 1IA100 1 ND Antibodies were pre-incubated with a set concentration ofligand, usually an EC70-80 for activating calcium mobilization at theBradykinin B2 Receptor. The antibody-ligand mixture was added to MRC5Fetal Lung Fibroblasts (ATCC CCL-171) pre-loaded with a calcium sensingdye (Fluo-4AM or Fluo-8AM) on the Hamamatsu FDSS6000 instrument, andcalcium mobilization was monitored. Data was exported as a max-minrelative fluorescence of the biological response, and IC50 for liganddepletion was calculated using sigmoidal curve fit in Graph Pad PrismV4.03. Data reported as molar ratio for ligand depletion by the antibodyto standardize the different concentration of ligand that was used forthe various experiments. Molar Ratio for ligand depletion = [IC50 ofAntibody]/[Ligand] SD = Standard Deviation; ND = not determined; IA100 =Inactive at 100 nM; IA150 = Inactive at 150 nM; IA300 = Inactive at 300nM; IA400 = Inactive at 400 nM; IA600 = Inactive at 600 nM

EXAMPLE 5 Engineering of F151: Humanization, Stabilization and Mutationof Unwanted Sequence Motifs 1. Humanization

The humanization protocol used has been described in PCT/US08/74381(US20110027266), herein incorporated by reference in its entirety. Thevariable light (VL) and variable heavy (VH) sequences of murine F151were used to build a homology model of anti-DAKD/KD F151 LC and HC inMolecular Operating Environment (MOE; v. 2009.10; Chemical ComputingGroup). The following templates were used: light chain framework—1SBS(93% identity in the framework regions), heavy chain framework—2VXT (84%identity in the framework regions), L1-1LVE (93% identity), L2-1 EEU(100% identity), L3-2R56 (93% identity), H1-1NJ9 (95% identity), H2-2VXU(76% identity) and H3-1HIL (49% identity). Templates were available atthe RCSB Protein Data Bank found on the world wide web at rcsb.org, awebsite managed by Rutgers and the University of California San Diego(Berman, H. M; Westbrook J.; Feng. Z.; Gilliland, G.; Bhat, T. N.;Weissig, H.; Shindyalov, I. N.; Bourne, P. E. The Protein Data Bank,Nucleic Acids Research, 2000, 28, 235-242.). The homology model wassubsequently energy minimized using the standard procedures implementedin MOE. A molecular dynamics (MD) simulation of the minimized 3Dhomology model of the murine F151 was subsequently performed, withconstraints on the protein backbone at 500 K temperature for 1.1nanoseconds (ns) in Generalized Born implicit solvent. Ten diverseconformations were extracted from this first MD run every 100picoseconds (ps) for the last 1 ns. These diverse conformations werethen each submitted to a MD simulation, with no constraints on theprotein backbone and at 300 K temperature, for 2.3 ns. For each of the10 MD runs, the last 2,000 snapshots, one every ps, from the MDtrajectory were then used to calculate, for each murine F151 amino acid,its root mean square deviations (rmsd) compared to a reference medoidposition. By comparing the average rmsd on the 10 separate MD runs of agiven amino-acid to the overall average rmsd of all F151 murineamino-acids, one decides if the amino-acid is flexible enough, as seenduring the MD to be considered as likely to interact with T-cellreceptors and responsible for activation of the immune response. 62amino-acids were identified as flexible in the murine F151 antibody,excluding the CDR and its immediate 5 Å vicinity.

The motion of the 28 most flexible murine F151 amino acids, during the20 ns (10×2 ns), were then compared to the motion of the correspondingflexible amino-acids of 49 human germline homology models, for each ofwhich were run the 10×2 ns MD simulations. The 49 human germline modelswere built by systematically combining the 7 most common human germlinelight chains (vk1, vk2, vk3, vk4, vlambda1, vlambda2, vlambda3) and 7most common human germline heavy chains (vh1a, vh1b, vh2, vh3, vh4, vh5,vh6). The vk1-vh1b human germline antibody showed 0.80 4D similarity ofits flexible amino-acids compared to the flexible amino-acids of themurine F151 antibody; the vk1-vh1b germline antibody was therefore usedto humanize F151 antibody focusing on the flexible amino-acids. For thepair wise amino-acid association between murine F151 vk1-vh1bamino-acids, the 2 sequences were aligned based on the optimal 3Dsuperposition of the alpha carbons of the 2 corresponding homologymodels (see FIG. 15 for an alignment of F151 LC and F151 HC with vk1 andvh1b, respectively).

2. Stabilization

Two approaches were used to improve the stability of the antibody.

a) Knowledge-Based Approach

The amino acids of the light and heavy chains with low frequency ofoccurrence vs. their respective canonical sequences, excluding the CDRs,were proposed to be mutated into the most frequently found amino acids(ΔΔGth>0.5 kcal/mol; E. Monsellier, H. Bedouelle. J. Mol. Biol. 362,2006, p. 580-593). This first list of consensus mutations for the lightchain (LC) and heavy chain (HC) was restricted to the amino acids foundin the closest human germline (vk1-vh1b). Suggested changes in theimmediate vicinity of the CDRs (5 Angstroms “Vernier” zone, J. Mol.Biol. 224, 1992, p. 487-499) were removed from consideration. Thisresulted in 5 stabilizing mutations in the LC (see Table 19) and 4stabilizing mutations in the HC (see Table 20). Other criteria weretaken into account to consider these mutations for potentiallystabilizing the anti-DAKD/KD F151 antibody. These criteria were afavorable change of hydropathy at the surface or a molecular mechanicsbased predicted stabilization of the mutant. Also, additionalstabilizing mutations reported to be successful in the literature (E.Monsellier & H. Bedouelle, J. Mol. Biol., 362, 2006, p. 580-593; B. J.Steipe et al. J. Mol. Biol, 1994, 240, 188-192) were considered (seeTables 16-22). One of these changes was incorporated as a stabilizingmutation (D89E) in sequences HC2a, HC2b and HC2c below. Anothersuggested change (Q62E) was incorporated in variant HC2b.

b) 3D and MD-Based Approaches

3D and MD-based approaches have been previously reported (Seco J, LuqueF J, Barril X., J Med Chem. 2009 Apr. 23; 52(8):2363-71; Malin Jonssonet al., J. Phys. Chem. B 2003, 107, 5511-5518). Hydrophobic regions ofthe antibody were explicitly identified by analyzing the moleculardynamics simulation of the Fab in a binary solvent (20% isopropanol inwater, 20 ns production simulation). Lysine mutations were thenintroduced in the vicinity of these regions as an attempt to prevent theaggregation. Additional analysis using a hydrophobic surface map withinSchrodinger's maestro software (v. 8.5.207) was completed. Using acombination of these two techniques, 2 Lys mutations, 1 in the heavychain and 1 in the light chain, are suggested.

3. Humanization by Grafting

Humanization using grafting grafting techniques has previously beenreported (Peter T. Jones, Paul H. Dear, Jefferson Foote, Michael S.Neuberger & Greg Winter Nature, 1986, 321, 522-525). The humanizationprocess which was used started by identifying the closest humangermlines to anti-DAKD/KD light and heavy chains. This is done byperforming a BLAST search vs. all the human germlines which weresystematically enumerated (all possible combinations of the V & Jdomains for the kappa and lambda chains; V, D and J domains for theheavy chains).

The following closest human germlines were identified with 83% and 62%sequence identity to anti-DAKD/KD F151 light chains (LC) and heavychains (HC), respectively (see FIG. 16). Using the internal VBASEgermline, the light chain is found to be close to V IV-B3 (˜83%identity) locus and the heavy chain close to 1-08 & 1-18 (˜62% identity)locus of the VH1 sub-family. CDR regions (as defined by MOE), andVernier regions (as defined in Foote & Winter, J. Mol. Biol., 1992, 224,487-499) are indicated in boldface The humanizing mutations inunderlining were obtained by performing a pairwise comparison of the 2aligned sequences, excluding the CDR & Vernier zone residues as definedabove. In another variant of the humanization, only the CDRs wereexcluded in the comparison.

4. Mutation of Unwanted Sequence MOTIFS

The following motifs of sequences were considered: Asp-Pro (acid labilebond), Asn-X-Ser/Thr (glycosylation, X=any amino-acid but Pro),Asp-Gly/Ser/Thr (succinimide/iso-asp formation in flexible regions),Asn-Gly/His/Ser/Ala/Cys (exposed deamidation sites), and Met (oxidationin exposed areas). Among other criteria, the VL & VH domains of murineF151 was selected from other murine antibodies because murine F151 didnot have exposed unwanted sequence motifs, but they are introduced insome humanized variants.

LC3a, LC3b, HC3a and HC3b each have potentially problematic succinimidesites that were identified. These sites were not modified in theproposed sequences as the residues involved are potentially involved inH-bond network (visual inspection of the homology model). Thesepositions are also found in a number of other antibody structures.Additionally, in both HC3a and HC3b, a strict humanization by graftingwould include a substitution of Ser115 to Met. This Methionine isexposed. A substitution to Leucine at this position is suggested as ahumanizing mutation as it is a common residue among many close humangermline sequences.

The resulting humanized sequences were blasted for sequence similarityagainst the International Epitope Database (IEDB) database (found on theworld wide web at immuneepitope.com; version June 2009; Vita R, ZarebskiL, Greenbaum J A, Emami H, Hoof I, Salimi N, Damle R, Sette A, Peters B.The immune epitope database 2.0. Nucleic Acids Res. 2010 January; 38(Database issue):D854-62. Epub 2009 Nov. 11) to ensure that none of thesequences contain any known human B- or T-cell epitopes (sequenceidentity of 70% used as cut-off for the results obtained through BLASTsearch and considering only the results from human species).

5. Original Sequences of Murine F151 Variable Domains

CDRs are highlighted in bold and Vernier regions (as defined in Foote &Winter, J. Mol. Biol., 1992, 224, 487-499) are underlined.

Light Chain (SEQ ID NO: 26) DIVMSQSPSS LAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSP KPLIY WASTRESGVPDRFTGSGSGTDFTLT ISSVKAEDLA IYYCQQYYSYPWTFGGGTKLEIK Germinality index = 83% with Z46615_1_V_X67858_1_J [V IV-B3]Heavy Chain (SEQ ID NO: 19): EIQLQQSGPELVKPGTSVKVSCKASG YSFT DYNIYWVKQSHGKSLEWIG Y FDPYNGNTGYNQKFRGKATLTVDKSSSTAF MHLSSLTSDDSAVYYCANYYRYDDHAMDY WGQGTSVTVSS Germinality index = 62% withZ12316_1_VX97051_4_D_X97051_5_J [VH1 1-18]

6. Engineered Sequences a) Background

5 versions for the light chain (LC1, LC2a, LC2b, LC3a, and LC3b) and 5versions of the heavy chain (HC1, HC2a, HC2b, HC3a, and HC3b) wereproposed.

LC1 contains 5 humanizing mutations identified using the 4D humanizationprotocol. LC2a introduced an additional 5 stabilizing mutations. LC2badded 1 Lysine mutations to help prevent aggregation. LC3a contains 15mutations derived from grafting to the closest human germline sequenceand retaining the murine CDR and Vernier zone residues. LC3b contained16 mutations derived from CDR-grafting with one additional humanizingmutation.

HC1 has 6 humanizing mutations identified by the in-house protocol. HC2aintroduced 5 additional stabilizing mutations while HC2b contains 6additional stabilizing mutations as compared to HC1. HC2c contains 1 Lysmutation, in addition to the stabilizing mutations of HC2a, to helpprevent aggregation. HC3a contains 19 mutations derived from grafting tothe closest human germline sequence and retaining the murine CDR andVernier zone residues. HC3b contains 25 mutations derived from CDRgrafting.

6 combinations in total were proposed (summarized in Table 16):

-   -   LC1×HC1 (mutations addressing humanization only)    -   LC2a×HC2a (mutations addressing humanization and stabilization)    -   LC2a×HC2b (mutations addressing humanization and stabilization)    -   LC2b×HC2c (mutations addressing humanization, stabilization and        “anti-aggregation”)    -   LC3a×HC3a (mutations addressing mostly humanization by        grafting+Vernier)    -   LC3b×HC3b (mutations addressing humanization by grafting)

TABLE 16 Summary of the 6 LCxHC combinations proposed (LC2b) (LC3a)(LC2a) Humanizing + Grafting (LC1) Humanizing + stabilizing + anti- WithVernier (LC3b) Humanizing stabilizing aggregation Regions Grafting (HC1)X Humanizing (HC2a) X Humanizing + stabilizing (HC2b) X Humanizing +stabilizing (HC2c) X Humanizing + stabilizing + “anti- aggregation”(HC3a) X grafting (HC3b) X grafting

TABLE 17 Mutations of the 5 LC variants of the anti-DAKD/KD F151antibody (LC2b) Humanizing + (LC3a) Light Light (LC2a) stabilizingGrafting (LC3b) Chain Chain (LC1) Humanizing + mutations + CDRs +Grafting Sequential Kabat Humanizing stabilizing anti-aggregationVernier CDRs numbering Numbering mutations mutations mutations residuesonly Ser5 Ser5 Thr Thr Thr Thr Ser9 Ser9 Asp Asp Ala12 Ala12 Ser SerVal13 Val13 Ala Ala Ala Val15 Val15 Leu Leu Glu17 Glu17 Asp Asp AspLys18 Lys18 Arg Arg Arg Arg Arg Val19 Val19 Ala Ala Met21 Met21 Ile IleIle Ile Ser22 Ser22 Asn Asn Gln48 Gln42 Lys Lys Lys Ser49 Ser43 Pro ProPro52 Pro46 Leu Thr69 Thr63 Ser Ser Ser Ser Val84 Val78 Leu Leu Lys85Lys79 Gln Gln Gln Gln Gln Leu89 Leu83 Lys Val Val Ile91 Ile85 Thr ThrVal Val Gly106 Gly100 Gln Gln Leu110 Leu104 Val Val Mutations: 5 10 1115 16

TABLE 18 Mutations of the 6 HC variants of the anti-DAKD/KD F151antibody (HC2c) humanizing + (HC3a) Heavy Heavy (HC2a) (HC2b)stabilizing Grafting (HC3b) Chain Chain (HC1) Humanizing + Humanizingmutations + CDRs + Grafting Sequential Kabat Humanizing stabilizingstabilizing antiaggregation Vernier CDRs numbering numbering mutationsmutations mutations mutations residue only Glu1 Glu1 Gln Gln Gln Gln GlnIle2 Ile2 Val Gln5 Gln5 Val Val Val Val Val Val Pro9 Pro9 Ala Ala AlaAla Ala Leu11 Leu11 Val Val Val Val Val Val Val12 Val12 Lys Lys Lys LysLys Lys Thr16 Thr16 Ala Ala Ala Ala Ala Ala Lys38 Lys38 Arg Arg Ser40Ser40 Ala Ala His41 His41 Pro Pro Pro Pro Pro Pro Lys43 Lys43 Gln GlnSer44 Ser44 Gly Gly Gly Gly Gly Ile48 Ile48 Met Gln62 Gln61 Glu Lys67Lys66 Arg Arg Ala68 Ala67 Val Leu70 Leu69 Met Val72 Val71 Thr Lys74Lys73 Thr Ser76 Ser75 Thr Thr Phe80 Phe79 Tyr Tyr Tyr Tyr Tyr His82His81 Glu Glu Ser84 Ser82A Arg Arg Leu 86 Leu82C Lys Thr87 Thr83 Arg ArgAsp89 Asp85 Glu Glu Glu Glu Asp90 Asp86 Glu Glu Glu Ser91 Ser87 Thr ThrSer115 Ser108 Leu Leu Mutations: 6 11 12 12 19 25

a) Engineered Light Chain Sequences:

No potentially problematic known T-cell or B-cell epitopes were found inall the variants proposed.

LC1 (SEQ ID NO:27), humanizing mutations are underlined, CDRs andvernier zones are in bold:

DIVMSQSPSSLAASVGDRVTMSCKSSQSLLYSSNQKNYLAWYQQKPGKSP KPLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAIYYCQQYYSYPWTFGGGTKLEIK

LC2a (SEQ ID NO:28), humanizing mutations are underlined, CDRs andvernier zones are in bold, stabilization mutations are in italics (T atposition 5, S at position 12, I at position 21, S at position 69, T atposition 91 shown below) :

DIVM TQSPSSLS ASVGDRVTISCKSSQSLLYSSNQKNYLA WYQQKPGKSPKPLIYWASTRESGVPDRFSGSGSGTDFTLT ISSVQAEDLA TYYCQQYYSYPWTFGGGTKLEIK

LC2b (SEQ ID NO:29) humanizing mutations are underlined, CDRs andvernier zones are in bold, stabilization mutations are in italics (T atposition 5, S at position 12, I at position 21, S at position 69, T atposition 91 shown below) and an anti-aggregation mutation is K atposition 89:

DIVM TQSPSSLS ASVGDRVTISCKSSQSLLYSSNQKNYLA WYQQKPGKSPKPLIYWASTRESGVPDRFSGSGSGTDFTLT ISSVQAEDKA TYYCQQYYSYPWTFGGGTKLEIK

LC3a (SEQ ID NO:30), grafted mutations shown in underline and CDRs andvernier zones shown in bold:

DIVM TQSPDSLAVSLGERATIN CKSSQSLLYSSNQKNYLA WYQQKPGQPPKPLIYWASTRESGVRDRFSGSGSGTDFTLT ISSLQAEDVAVYYCQQYYSYPWTFGQGTKVEIK

LC3b (SEQ ID NO:31), grafted mutations shown in underline and CDRs andvernier zones shown in bold:

DIVM TQSPDSLAVSLGERATINCKSSQSLLYSSNQKNYLA WYQQKPGQPPKL LIYWASTRESGVPDRFSGSGSGTDFTLT ISSLQAEDVAVYYCQQYYSYPWTFGQGTKVEIK

Note that L at position 52 is a vernier residue that is mutated tohuman.

c) Engineered Heavy Chain Sequences

HC1 (SEQ ID NO:20), humanizing mutations are underlined, CDRs andvernier zones are in bold:

EIQLVQSGPEVKKPGASVKVSCKASGYSFTDYNIYWVKQSPGKSLEWIGYFDPYNGNTGYNQKFRGKATLTVDKSSSTAFMHLSSLTSEDSAVYYCANYYRYDDHAMDYWGQGTSVTVSS

HC2a (SEQ ID NO:21), humanizing mutations are underlined, CDRs andvernier zones are in bold, stabilization mutations are in italics (Q atposition 1, A at position 9, G at position 44, Y at position 80 and E atposition 90 shown below):

Q IQLVQSGAEVKKPGASVKVSCKASGYSFTDYNIYWVKQSPGKGLEWIGYFDPYNGNTGYNQKFRGKATLTVDKSSSTA Y MHLSSLTSEESAVYYCANYYRYDDHAMDYWGQGTSVTVSS

HC2b (SEQ ID NO:22), humanizing mutations are underlined, CDRs andvernier zones are in bold, stabilization mutations are in italics (Q atposition 1, A at position 9, G at position 44, E at position 62, Y atposition 80 and E at position 90 shown below):

Q IQLVQSGAEVKKPGASVKVSCKASGYSFTDYNIYWVKQS PGKGLEWIGYFDPYNGNTGYN EKFRGKATLTVDKSSSTA Y MHLSSLTSE ESAVYYCANYYRYDDHAMDYWGQGTSVTVSS

No human epitopes were identified for sequence HC2b in IEDB database.

HC2c (SEQ ID NO:23), humanizing mutations are underlined, CDRs andvernier zones are in bold, stabilization mutations are in italics (Q atposition 1, A at position 9, G at position 44, Y at position 80 and E atposition 90 shown below) and an anti-aggregation mutation at K atposition 86:

Q IQLVQSGAEVKKPGASVKVSCKASGYSFTDYNIYWVKQSPGKGLEWIGYFDPYNGNTGYNQKFRGKATLTVDKSSSTA Y MHLSSKTSEESAVYYCANYYRYDDHAMDYWGQGTSVTVSS

HC3a (SEQ ID NO:24), grafted mutations shown in underline and CDRs andvernier zones shown in bold:

Q IQLVQSGAEVKKPGASVKVSCKASGYSFTDYNIYWVRQA PGQGLEWIGYFDPYNGNTGYNQKFRGRATLTVDKSTSTA Y MELRSLRSDDTAVYYCANYYRYDDHAMDYWGQGTLVTVSS

LC3b (SEQ ID NO:25), grafted mutations shown in underline and CDRs andvernier zones shown in bold:

QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYNIYWVRQA PGQGLEW M GYFDPYNGNTGYNQKFRGRVTMTTDTSTSTA Y MELRSLRSDDTAVYYCANYYRYDDHAMDYW GQGTLVTVSS

Note that the following Vernier Residue are mutated to human: V atposition 2, M at position 48, V at position 68, M at position 70 and Tat position 74.

No human epitopes were identified for sequence HC3b in IEDB database.

HC3b germinality index=83% withZ12316_(—)1_V_J00235_(—)1_D_U42590_(—)1_J [1-18/DP-14].

TABLE 19 Stabilizing Changes Proposed in Light Chain Residue ProposedChange Calculated Gth Accept Change Ser-5 Thr 2.32286 Yes Ala-12 Ser0.75228 Yes Met-21 Ile 0.768959 Yes Pro-52 Leu 1.70059 No - Vernierregion Thr-69 Ser 1.10843 Yes Lys-86 Glu 2.00115 No - changed to Glnduring humanization Ile-91 Thr 1.27255 Yes

TABLE 20 Stabilizing Changes Proposed in Heavy Chain Residue ProposedChange Calculated Gth Accept Change Glu-1 Gln 0.562423 Yes Ile-2 Val2.15882 No - Vernier region Pro-9 Ala 0.505324 Yes Thr-16 Ala 1.50552Already changed to Ala in humanization Val-20 Leu 2.21586 No - not ingermline sequence Ser-40 Arg 1.03643 No - not in germline sequenceHis-41 Pro 1.67738 Already changed to Pro in humanization Ser-44 Gly1.5068 Yes Gln-62 Glu 0.74934 No - not in germline sequence Arg-65 Lys2.32314 No - not in germline sequence Phe-80 Tyr 1.30935 Yes His-82 Gln2.24674 No - not in germline sequence Asp-89 Glu 1.65409 Already changedto Glu in humanization Asn-98 Arg 3.65643 No - Vernier region

TABLE 21 Combinations of stabilizing mutations evaluated Additionalchanges Combination* suggested Accept Change L1 (46->P & 48->Q) K48->QNo - K48 humanizing mutation L2 (51->K) None - already None K51 L3(80->T) None - already None T80 L4 (82->S) None - already None S82 L5(90->A, 91->T) None - already None A90, T91 suggested above (Table 1) H1(15->G) None - already None G15 H2 (62->E, 63->K, 64->F) Q62->E, Yes -considered in HC2b already K63 and F64 H3 (87->T, 88->S, 89->D) D89->E,Yes - potential salt bridges already with K63 and K43 T87 and S88 S1 (L1& L5) K48->Q No - K48 humanizing mutation S2 (H1 & H3) D89->E No (seeH3) *Note: Sequential numbering used to refer to residues

TABLE 22 Potential Stabilizing Mutations Light Chain Residue* Additionalchanges suggested Accept Change 15->L V15->L No - V15 in Vk1 germline96->Q None - already Q96 None 38->Y None - already Y38 None 112->INone - already I112 None 69->S G69->S No - G69 is in Vernier Region21->I M21->I Already changed (see Table 19) *Note: Sequential numberingused to refer to residues

EXAMPLE 6 Characterization of Humanization Variants

Based on the in silico modeling presented in Table 16, the variableregion of the light chain (VL) and heavy chain (VH) DNA of humanizedF151 were codon optimized for HEK293 expression and gene synthesized byGeneArt (subsidiary of Life Technologies). The synthesized DNA fragmentswere cloned into the constant region of the light chain (CL) encodingvectors, pFF0362 (A. Human Kappa LC vector) at ApaLI/BsiWI sites and theconstant regions of the heavy chain (CH1, CH2 and CH3) encoding vectors,pFF0363 (B. Human IgG1 HC vector) at ApaLI/ApaI sites respectively. Theresulted plasmids pFF0460 containing the full sequence of LC and pFF0466containing the full length of HC of humanized F151 variants wereco-transfected and transiently expressed in FreeStyle™ 293 ExpressionSystem (Invitrogen/Life Technologies, catalog no. K9000-01).

The six humanized variants shown in Table 16 were characterized byvarious parameters such as binding kinetics (discussed above) as well aschemical and physical properties such as thermostability that areroutinely used in the art.

The characterization was done in two tiers. Tier I included differentialscanning calorimetry (DSC) shown in Table 24 and FIG. 2. Briefly, forthe DCS experiments, the antibodies were dialyzed againstphosphate-buffered saline solution. Antibody concentrations weremeasured by UV absorbance. The antibodies were diluted to 1 mg/mL usingPBS. Scans were performed using a Calorimetry Sciences Corporation N-DSCII instrument using a 0.3268 mL capillary cell with PBS in the referencecell. The scan rate was 2° C./min and the samples were scanned from 20°C. to 100° C.

All variants, except for HC3b/LC3b showed comparable binding affinitiesto the parental antibody. Variant HC3a/LC3a was selected over the othervariants based on other physiochemical properties such as SEC data,stability and lack of aggregation (see Tables 23-25).

TABLE 23 Comparison of Kinetics of the Humanized F151 Variants HC1/LC1HC2a/LC2a Ka(1/Ms) Kd(1/s) KD(M) Ka(1/Ms) Kd(1/s) KD(M) DAKD-b 4.16E+056.00E−06 1.45E−11 6.66E+05 1.22E−05 1.83E−11 KD-b 4.24E+05 1.74E−073.94E−13 7.03E+05 6.12E−06 8.71E−12 DAKLP-b 5.00E+05 7.96E−06 1.60E−114.10E+05 5.67E−06 1.38E−11 KLP-b 4.81E+05 2.67E−06 5.54E−12 6.15E+052.68E−05 4.34E−11 HC2b/LC2a HC2c/LC2b Ka(1/Ms) Kd(1/s) KD(M) Ka(1/Ms)Kd(1/s) KD(M) DAKD-b 4.17E+05 1.05E−05 2.57E−11 4.81E+05 4.34E−059.01E−11 KD-b 3.75E+05 1.66E−06 4.72E−12 5.64E+05 9.08E−06 1.74E−11DAKLP-b 4.46E+05 1.30E−05 2.97E−11 9.03E+05 1.10E−05 1.21E−11 KLP-b4.01E+05 2.20E−06 5.76E−12 5.16E+05 1.02E−05 1.98E−11 HC3a/LC3aHC3b/LC3b Ka(1/Ms) Kd(1/s) KD(M) Ka(1/Ms) Kd(1/s) KD(M) DAKD-b 5.06E+051.28E−05 2.53E−11 3.85E+05 5.15E−05 1.35E−10 KD-b 4.27E+05 2.95E−066.78E−12 2.51E+05 3.02E−06 1.44E−11 DAKLP-b 4.65E+05 1.42E−05 3.05E−117.04E+04 2.76E−03 4.05E−08 KLP-b 5.02E+05 5.43E−07 1.06E−12 5.39E+052.72E−04 5.26E−10 For comparison: Ka(1/Ms) of mF151 was 7.84E+05 forDAKD-b, 8.30E+05 for KD-b, 1.81E+06 for DAKLP-b, and 1.12E+06 for KLP-b

TABLE 24 Tier 1 comparison of humanization variants Test ProteinFunctional Conc. Potency Ligand (mg/ml) Purity Stability Assay AffinityMethod UV 1D-gel DSC (Tm ° C.) FDSS Assay Spectroscopy (reducing & (Tmof parental without Variant (A280) non-reducing) SEC F. 151 = 73° C.)FDSS Assay preincubation Biacore Binding HC1/ 1.69 No ≈5% 81.6(M) nMpotency at DAKD, Active at DAKD and Kon 10E5, koffless than or LC1Ag/Deg Ag 70.0(m) sub nM potency at KD, KD comparable among equal to10E−6 HC2a/ 2.01 No No Ag 75.0(M) comparable among 5 variants comparableamong 5 variants LC2a Ag/Deg 82.5(m) 5 variants HC2b/ 1.83 No No Ag74.6(M) LC2a Ag/Deg 83.0(m) HC2c/ 0.68 No No Ag 71.0(M) LC2b Ag/Deg64.0(m) 82.5(m) unstable HC3a/ 1.71 No No Ag 82.3(M) LC3a Ag/Deg 71.8(m)Most Stable HC3b/ 1.47 No No Ag 79.4(M) Lost potency to Decreasedaffinity LC3b Ag/Deg 71.4(m) KLP & DAKLP to KLP & DAKLP Abbreviations:Ag = aggregation, Deg = degradation

TABLE 25 Tier 2 comparison of humanization variants N-terminalIntactness sequence Confirmation confirmation Thermostability Stability(LC, HC) N-terminal Variant 1D-gel SEC Biacore SEC LC-MS sequencing HC1/No No 45 C. No LC(+1 Da off) N terminal of LC LC1 Ag/Deg Ag/Deg slightlyAg/Deg HC(+1 Da off) & HC intact faster off G0 dominant rate than 4 C.HC2a/ No No 45 C. No LC(spot on) N terminal of LC LC2a Ag/Deg Ag/Degslightly Ag/Deg HC(spot on) & HC intact faster off G0 dominant rate than4 C. HC2b/ No No 45 C. No LC(+1 Da off) N terminal of LC LC2a Ag/DegAg/Deg slightly Ag/Deg HC(+1 Da off) & HC intact faster off G0 dominantrate than 4 C. HC2c/ X X X X X X LC2b HC3a/ No No 45 C. No LC(−2 Da off)N terminal of LC LC3a Ag/Deg Ag/Deg slightly Ag/Deg HC(−2 Da off) & HCintact faster off G0 dominant rate than 4 C. HC3b/ X X X X X X LC3bThermostability = Incubation at 4° C. (control) and 45° C. for 3 days;1D-gel was under non-reducing conditions; Stability = 2 cycles offreeze/thaw; LC-MS = reduced and reduced/deglycosylation Abbreviations:Ag = aggregation, Deg = degradation, X = no data presented

TABLE 26 Comparison of Parental F151 and humanized variant F151 (HC3a/LC3a) Variable Heavy Chain Variable Light Chain Parental F151 GeneGAGATCCAGCTGCAGCAGTCTGGACCTGAGCTG GACATTGTGATGTCACAGTCTCCATCCTGTGAAGCCTGGGACTTCAGTGAAGGTGTCCTGC CCCTAGCTGTGTCAGTTGGAGAGAAGGTAAGGCTTCTGGTTACTCATTCACTGACTACAAC TACTATGAGCTGCAAGTCCAGTCAGAGCATCTACTGGGTGAAACAGAGCCATGGAAAGAGC CTTTTATATAGTAGCAATCAAAAGAACTCTTGAGTGGATTGGATATTTTGATCCTTACAAT ACTTGGCCTGGTACCAGCAGAAACCAGGGGTAATACTGGCTACAACCAGAAGTTCAGGGGC GCAGTCTCCTAAACCGCTGATTTACTGGAAGGCCACATTGACTGTTGACAAGTCCTCCAGC GCATCCACTAGGGAATCTGGGGTCCCTGACAGCCTTCATGCATCTCAGCAGCCTGACATCT ATCGCTTCACAGGCAGTGGATCTGGGACGATGACTCTGCAGTCTATTACTGTGCAAACTAC AGATTTCACTCTCACCATCAGCAGTGTGTATAGGTATGACGACCATGCTATGGACTATTGG AAGGCTGAAGACCTGGCAATTTATTACTGGTCAAGGAACCTCAGTCACCGTCTCCTCA GTCAGCAATATTATAGCTATCCGTGGAC(SEQ ID NO: 127) GTTCGGTGGAGGCACCAAGCTGGAAATC AAA (SEQ ID NO: 128)Protein EIQLQQSGPELVKPGTSVKVSCKASGYSFTDYN DIVMSQSPSSLAVSVGEKVTMSC KSSQSLIY WVKQSHGKSLEWIG YFDPYNGNTGYNQKFRG LYSSNQKNYLA WYQQKPGQSPKPLIY WAKATLTVDKSSSTAFMHLSSLTSDDSAVYYCANY STRES GVPDRFTGSGSGTDFTLTISSVKYRYDDHAMDY WGQGTSVTVSS AEDLAIYYC QQYYSYPWT FGGGTKLEIK (SEQ ID NO: 19)(SEQ ID NO: 26) Humanized F151 (HC3a/LC3a) GeneCAGATTCAGCTGGTGCAGTCTGGCGCCGAAGTG GACATCGTGATGACCCAGAGCCCCGACAAAGAAACCTGGCGCCAGCGTGAAGGTGTCCTGC GCCTGGCCGTGTCTCTGGGCGAGCGGGCAAGGCCAGCGGCTACAGCTTCACCGACTACAAC CACCATCAACTGCAAGAGCAGCCAGAGCATCTACTGGGTCCGACAGGCTCCAGGCCAGGGA CTGCTGTACTCTAGCAACCAGAAGAACTCTGGAATGGATCGGCTACTTCGACCCCTACAAC ACCTGGCCTGGTATCAGCAGAAGCCCGGGGCAACACCGGCTACAACCAGAAGTTCCGGGGC CCAGCCCCCCAAGCCCCTGATCTACTGGAGAGCCACCCTGACCGTGGACAAGAGCACCAGC GCCAGCACCCGCGAGAGCGGCGTGCCCGACCGCCTACATGGAACTGCGGAGCCTGAGAAGC ATAGATTTTCCGGCAGCGGCTCCGGCACGACGACACCGCCGTGTACTACTGCGCCAACTAC CGACTTCACCCTGACCATCAGCAGCCTGTACAGATACGACGACCACGCCATGGACTACTGG CAGGCCGAGGACGTGGCCGTGTACTACTGGCCAGGGCACCCTGGTCACCGTGTCCTCT GCCAGCAGTACTACAGCTACCCCTGGAC(SEQ ID NO: 129) CTTCGGCCAGGGCACCAAGGTGGAAATC AAG (SEQ ID NO: 130)Protein QIQLVQSGAEVKKPGASVKVSCKASGYSFTDYN DIVMTQSPDSLAVSLGERATINC KSSQSIY WVRQAPGQGLEWIG YFDPYNGNTGYNQKFRG LLYSSNQKNYLA WYQQKPGQPPKPLIY WRATLTVDKSTSTAYMELRSLRSDDTAVYYCANY ASTRES GVPDRFSGSGSGTDFTLTISSLYRYDDHAMDY WGQGTLVTVSS QAEDVAVYYC QQYYSYPWT FGQGTKVEI (SEQ ID NO: 24) K(SEQ ID NO: 30) Single underscore = CDR region; double underscore =signature amino acids for identifying CDRs

For alignment of light and heavy chains of parental F151 to humanizedF151 variant (HC3a/LC3a), see FIG. 3.

EXAMPLE 7 Crystal Structure of Humanized Antibody F151 against BRK1Ligand Kallidan and des-arg¹⁰-Kallidin

The crystal structures of humanized F151 (HC3a/LC3a) Fab bound tokallidan or des-arg¹⁰-kallidin was determined and the molecularinteractions analyzed.

Kallidin powder was purchased from Phoenix Pharmaceuticals (Cat. No.009-37). For Fab protein generation, the DNA of heavy chain (HC) VHregion from humanized F151 HC3a was cloned into 6×His tagged CH1 vectorpFF0366 (“6×His” disclosed as SEQ ID NO: 137). The light chain (LC)plasmid used here was the same as of the original F151 LC3a used in F151humanization (see Example 5). The two plasmids were co-transfected intofree style HEK293 cells for Fab expression. The Fab protein was purifiedusing cobalt-resin, buffer exchanged to 50 mM MES pH6.0, 50 mM NaClbefore being concentrated to about 9 mg/m L. Purified F151 Fab proteinwas mixed with kallidin in a molar ratio of 1:2 and set up forcrystallization screening. Crystallization screening was done with awide range of conditions. The best crystal was observed under conditionB10, B12 and G10 of Hampton Research screening kit PEG/ION HT. Thecrystals were cryo-protected with 20% glycerol in well buffer and frozenfor diffraction data collection. The X-ray diffraction data for bothcomplexes were collected at Canadian Light Source, beamline CMCF-08ID.The Rmerge for the F151-KD complex is 8.9% and I/s(I)=20.2 , while thosefor the F151-DAKD are 7.7% and 18.5, respectively. The F151-KD structurewas solved by molecular replacement in Phaser using Fab coordinates fromPDB entry 3QOS, treating the V_(L)-V_(H) and C_(L)-C_(H)1 domains asindependent units. The structure was refined in autoBuster at 2.07 Åresolution in space group P2₁2₁2₁ to an Rfactor of 0.205 and an Rfree of0.228. The F151-DAKD structure was solved using the F151-KD coordinates.The structure was refined in autoBuster at 1.86 Å resolution in spacegroup P212121 to an Rfactor of 0.232 and an Rfree of 0.238.

The electron density maps shown in FIGS. 4 and 5 depict the binding ofkallidin (KD) and Des-Arg¹⁰-kallidin (DAKD) to the F151 Fab andunambiguously determine the positions of each amino acid. For kallidin,the electron density for the extreme C-terminal residue Arg¹⁰ is notpresent. This is in agreement with the observation that DAKD, which ismissing the C-terminal residue arginine (shown in Table 27 below), bindsequally well to F151 as KD. The 1050 values of F151 in theneutralization FDSS cellular assay towards KD and DAKD are 0.12 nM and0.09 nM, respectively. In both cases the electron density is weakertowards the C-termini of the peptides. Since Phe⁹ in KD has slightlybetter electron density than that in DAKD, it is possible that thepresence of the additional arginine at the C-terminus of KD stabilizesthe C-terminus of this peptide when binding to F151 although thisarginine itself is not stable enough to be observed by X-ray. Since thetwo structures are essentially identical (rms between KD and DAKD is0.139 for C atoms and 0.328 for all atoms), all the followingdiscussions are based on the F151-KD structure.

TABLE 27 A selected list of kinin peptides SEQ Peptide ID Sequence nameNO: 1 2 3 4 5 6 7 8 9 10 KD(Kallidin) 1Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg DAKD (des- 2Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe Arg¹⁰- Kallidin) KLP (rodent 3Arg-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg KD ortholog) BK 5Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg (Bradykinin)

KD is bound with its N-terminus buried in the interface between Fvsubunits of the light and heavy chains, as shown in FIG. 6. Theinterface between light and heavy chains are packed with aromatic aminoacids, including Tyr-L42, Tyr-L93, Tyr-L100, Trp_L102, Phe-L104 andTyr-H35, Trp-H47, Tyr-H50, Tyr-H99, Trp-H110, stabilizing each otherthrough stacking and hydrophobic interactions. Residues from each of theCDR's of light and heavy chains contribute to the binding. The residuesalong the light and heavy chains that are involved in interactions withKD as mapped on the CDRs are shown in FIGS. 7 and 8. CDR H3 of the heavychain is the longest loop and the one most frequently used in theinteractions with KD, forming a side cover for KD. The loop wasstabilized mostly through interactions with the other two CDRs, H1 andH2 of the heavy chain, namely, Salt bridge between Asp-H101 and Arg-H52(stabilizing H1 and H3), arene-H interaction between Tyr-H102 andTyr-H54 (stabilizing H2 and H3), H-bond between Asp-H108 and Tyr-H35 andH-bond between His-H105 and Tyr-L55 (stabilizing H3 and L2).

Comparing the KD interacting residues among the antibodies generated, itcan be seen that there is similarity among the antibodies, and some weremore related in use of particular amino acids for KD-interaction thanothers. For example, in the light chain F151, C63 and 122 use moresimilar amino acids in their CDRs to bind KD, while B21 and I54 weremore similar. In the heavy chain, F151 and C63 were surprisingly uniquefrom each other and from B21, I22 and I54. The latter three appear toform a group in similarity. C63 is particularly interesting in its heavychain, that the loop length in H2 and H3 are more different from allothers. Considering the Fab as a whole, B21 and I54 were most closelyrelated.

In the crystal structure, we found that KD is involved in systematichydrogen bond and hydrophobic interactions with the Fab. The N-terminusof KD is buried in the Fab and harbors more intensive interactions,while the C-terminus is essentially solvent exposed. Except for thefirst 4 residues (Lys-Arg-Pro-Pro) (SEQ ID NO: 132), the other residuesof KD gradually extend into the bulk solvent. The amidinium group ofLys1 sidechain is anchored by Glu-L61 (L: light chain) through saltbridges, while the amino terminal amino group Lys 1 forms a salt bridgewith Asp-H108 (H: heavy chain). The amidinium group of Lys1 sidechainalso hangs over the aromatic ring of Tyr-L55, involved in cationinteractions. Such intensive interactions involved with Lys1 tightlyanchor the amino terminus of KD in the Fab. This also accounts for theimportance of Lys1 in the binding of KD to F151. Without it (i.e.bradykinin), no detectable binding to hF151 or F151 can be measured.Like Lys1, Arg2 interacts with the Fab through a salt bridge. Theguanidium group of Arg2 interacts with the sidechain of Asp-H104. Thesidechain of Arg2 is also H-bonded with the mainchain carbonyl oxygen ofArg-H101. Also, the mainchain oxygen of Pro8 is H-bonded to thesidechain of Arg-H101. Tyr-H102 is half-way intercalated into Phe8 andPro9, involving in hydrophobic interactions with KD. In addition todirect interaction, numerous water-mediated H-bonds between KD and theFab are also seen. It is also interesting to notice that tyrosineresidues are most frequently used in the interaction compared to otheramino acids; 9 out of the 16 residues marked with asterisks in FIGS. 7and 8 are tyrosines. All the residues in F151 surrounding KD appear toplay a role in ligand binding, except for Asn-H33, which is close toPhe6 sidechain but incompatible in polarity and lack of other importantinteractions. Substitution with aromatic/hydrophobic residues, such Trpor Tyr to interaction with PheB appears to be a quick pick if affinitymaturation is considered. These two aromatic amino acids are in factseen in other antibodies (Trp in C63, and Tyr in B21, I22, I54). Table28 below provides a detailed analysis of 16 KD-interacting amino acidresidues marked in FIGS. 7 and 8 and sets forth functional substitutionsthat can be made in the CDR regions that should not disrupt antigenbinding.

TABLE 28 A list of amino acid residues found around the KD bindingpocket, and their roles in KD binding and potential functionalsubstitutions (light chain residues in grey-colored cells and heavychain residues in unshaded cells) Residue Role in KD Binding or CDRStabilization Functional Substitution Tyr-L31 Edge-on hydrophobicinteractions with Pro4 His to add an H-bond with amide N of Along withTyr-L38 and Tyr-L98 form three Pro5 orthogonal planes that surround the90 deg turn of other aromatic a.a, such as Trp, and KD at Pro4 PheTyr-L38 hydrophobic stacking with Pro4 His to add an H-bond withcarbonyl O Along with Tyr-L31 and Tyr-L98 form three of Arg2 orthogonalplanes that surround the 90 deg turn of KD at Pro4 Tyr-L55cation-interaction with amidinium ion of Lys1 Trp to pair with H105mutations of Gln, sidechain Asn, Glu or Asp (maintaining H-bond); H-bondwith His-H105; Tyr-L55--His-H105 pairing Other variants of His_H105 areTyr adds stabilization between L2 and H3 loops and Ser. Other aromatica.a., such as Trp, His and Phe Glu-L61 Forming key salt bridges withLys1 sidechain Asp, Gln, ASN (Asp is seen in B21 and I54 already, FIG.7) Tyr-L97 H-bond with amide N of Trp-L56 Aromatic a.a. such as Phe orHis (too Forming pocket for Arg1 extended sidechain tight space for Trp)Tyr-L98 Along with Tyr-L31 and Tyr-L38 form three Other aromatic a.a,such as Phe, Trp orthogonal planes that surround the 90 deg turn of orHis KD at Pro4 Tyr- Forming pocket surface for Pro3 Aromatic a.a., suchas Phe (better L100 Part of the aromatic a.a. cluster interface betweenhydrophobic interactions with Pro3) L/H chains, further includingTyr-L42, Tyr-L93, Other variants seen are Thr (in B21 Trp_L102, Phe-L104and Tyr-H35, Trp-H47, Tyr- and I54) and His (in I22) H50, Tyr-H99,Trp-H110 Partial stacking with Tyr-H50 Trp- Part of the aromatic a.a.cluster interface between Other aromatic a.a, such as Tyr (in L102 L/Hchains C63 and B21), Phe and His Stacking with Trp-H47 Other hydrophobicresidue, such as L (in I22) Asn-H33 Close to Phe6 sidechain butincompatible in Replace with aromatic/hydrophobic polarity, no otherroles seen either; can be a target a.a., such as Trp (seen in C63) orTyr for affinity maturation (seen inB21, I22, I54) Asp-H52 Salt bridgewith Arg-H101, stabilizing H1 and H3 Mutate as a pair with Arg-H101 toloops reversely charged a.a., such Arg- H52/Asp-H101, or a pair ofhydrophobic a.a. (Leu, Ile, Val, Met, Phe, Tyr, Trp, Ala) to form acluster with Phe6 of KD Tyr-H54 Close to Pro8 but no specificinteractions Mutated to negatively charged a.a. to Close to Arg-H101,but no charge interactions stabilize Arg-H101, such D or E (seen in B21,I22 and I54) or N or Q, also provide H-bond with carbonyl O of Pro8 (ALys in C63, which can be charge-reversed to Glu) Tyr-H99 Part ofaromatic interface between H and L chains Mutate to small aromatic a.a.except H-bond with Asn-H33 W, such as Phe and His Tight space Arg-H-bond with amide of Pro8 Mutate as a pair with Asp-H52 to H101supported by Asp-H52 (salt bridge) reversely charged a.a., such Arg-H52/Asp-H101, or a pair of hydrophobic a.a. to form a cluster with Phe6of KD Tyr- Half-way intercalating into Phe9 and Pro8, Phe can be better,Trp or His may be H102 hydrophobic interactions with KD OK too Asp- Keyresidue to salt-bridge with Arg2 Glu to maintain salt bridge with Arg2H104 Bigger a.a to fill the gap from Pro3, such as Tyr as seen in B21,I22 and I54 Asp- Key residue to salt bridge with N-term —NH3+ of KD GluH108 H-bond with Tyr-H33, stabilizing H3 loop Conserved residue! Not inCDR

Analysis of the conformational epitope of kallidin (KD) ordesArg10-Kallidin (DAKD) revealed that it adopts a “Pro4 kink”conformation. As depicted in FIG. 17, a hallmark of the “Pro 4 kink”conformation is a type II tight turn in the main chain polypeptidebackbone of KD or DAKD at Proline 4 (see Richardson J S. “The anatomyand taxonomy of protein structure.” Adv Protein Chem. 1981; 34:167-339,which is incorporated by reference herein). The “Pro 4 kink”conformation may further defined by all or substantially all of theremaining amino acids of KD (1-2 and 6-9) or DAKD adopting repeats of asigmoid shape which align the hydrophobic side chains in a spatiallystacking mode.

EXAMPLE 8 In vivo Pharmacology of Anti-BKR1-Ligand Antibodies in PainModels

The examples of the present invention illustrate the in vivo efficacy ofanti-BKR1 receptor-ligand antibodies in different preclinical models ofacute and chronic pain according to modified procedures described in (a)Saddi G M and Abbott F V., Pain (2000), 89:53-63; (b) Chen et al.,Molecular Pain (2010), 2:6-13 and (c) Bennett G J and Xie Y K., Pain(1988), 33:87-107.

Animals

Experiments were carried out using adult male OF1 mice (20-30 gr) forformalin studies and adult male C57BI/6J mice (25-30 gr) for both CFAand CCI studies. The mice were kept in a controlled temperature roomunder a 12-h light-dark cycle. Food and water were provided ad libitum.For all of the experiments, mice were acclimatized to the laboratoryroom for at least 2 hours before testing. No randomization was performedfor the studies. Experimenters performing the behavioral tests were notblind to treatment; however they were not aware of the study hypothesis.All procedures have been approved by the “Comité d′Expérimentation pourla Protection de I'Animal de Laboratoire” (Animal Care and UseCommittee) of sanofi-aventis recherche & développement and were carriedout in accordance with French legislation (Decree n°87-848-19 Oct. 1987-and decision -19 Apr. 1988) implementing European directive 86/609/EEC.

A. Formalin-Induced Acute Inflammatory Pain

The formalin test was used to measure nociceptive and inflammatory pain.Indeed, intraplantar injection of formalin induces an initial acutenociceptive behavioral response (0-12 minutes), followed by a secondinflammatory-mediated response (15-45 minutes), which is attributed tospinal cord excitability.

Formaldehyde (37%, Sigma) was diluted in saline (v/v) to obtain a 2.5%formaldehyde concentration (i.e. ≅6.25% formalin concentration). Micewere gently restrained and 20 μL of this solution was injectedsubcutaneously into the dorsal part of one hind paw. Behavioralresponses were scored immediately after formalin injection, then at 3minutes intervals over 45 minutes as follows: (0): normal weight bearingof the injected paw; (1): injected paw resting lightly on floor; (2):lifting-elevation of the injected paw; (3): licking or biting theinjected paw. Group sizes were 11-12 male OF1 mice.

Scores were plotted versus time and areas under the curves (AUC) werecalculated from the mean scores (±SEM) for both the early (0-12 min) andthe late (15-45 min) phases. Reversal of pain-like behaviors wasexpressed as change in AUC in %.

EE1 antibody inhibited the pain-like behavior in the late phase of theformalin test in male OF1 mice. EE1 antibody, when administeredintravenously 48 hours before intraplantar injection of formalin, showeda dose dependent reversal of the pain-like behavior only in the latephase with a Minimal Effective Dose (MED)=2.5 mg/kg, as depicted in FIG.9. Indeed, when administered at 2.5, 10 and 30 mg/kg, EE1 reversed thelate phase by 35±5%, 33±5% and 45±7%, respectively, as depicted in Table29.

In contrast, F151 weakly inhibits the pain-like behavior in the latephase of the formalin test when administered 48 hours beforeintraplantar injection of formalin. Indeed, when administered at 2.5 and10 mg/kg, F151 reversed the late phase by 15±7% and 21±5%, respectively,as depicted in Table 29.

TABLE 29 Effect of EE1 and F151 antibodies on formalin-induced pain-likebehavior in male OF1 mice Reversal of pain-like behavior Dose A.U.C. ±SEM (in %) ± SEM Group (mg/kg, i.v.) (15-45 min) (15-45 min) Isotype- 3063.6 ± 2.9  0 ± 5 control 1B7.11 (EE1) EE1 2.5 41.6 ± 3.4 (***) 35 ± 510 42.9 ± 2.9 (***) 33 ± 5 30 36.5 ± 4.3 (***) 45 ± 7 Isotype- 10 57.3 ±3  0 ± 5 control 1B7.11 (F151) F151 2.5 48.9 ± 3.8 (NS) 15 ± 7 10 45.0 ±2.8 (*) 21 ± 5 (*), p < 0.05; (***), p < 0.001: Student's t-test versusadequate control was used. NS: non significant

B. CFA (Complete Freund's Adjuvant)-Induced Chronic Inflammatory Pain

Chronic inflammation was induced under brief anesthesia (Isoflurane, 3%)by an intraplantar administration of 25 μL of Complete Freund's Adjuvant(CFA) containing 1 μg/μL heat-killed Mycobacterium tuberculosis inmineral oil and mannide monooleate (Sigma). Group sizes were 8 maleC57BI/6 mice.

EE1 antibody was administered intravenously 22 hours after intraplantarCFA injection at 2.5 and 30 mg/kg and mechanical and thermalhypersensitivities were assessed at Day 1 (D1), Day 4 (D4) and Day 7(D7) post-CFA intraplantar administration.

B1. Mechanical Hypersensitivity

Mechanical hypersensitivity was assessed by measuring the Frequency ofwithdrawal Response (FR, in %) following 10 applications of a 0.6 g VonFrey filament (Bioseb, France) onto the plantar surface of the injectedpaw.

To investigate the efficacy of EE1 antibody on pain-like behavior, wecalculated the reversal of mechanical hypersensitivity (in %) asfollows:

Percent reversals were calculated as(MeanFR-isotype-control_(postdose)−FR-Ipsi_(postdose))/(MeanFR-isotype-control_(postdose)−Mean FR-sham_(postdose))for each mouse.

At D1, D4 and D7 after intraplantar injection of CFA, a significantincrease of FR to the Von Frey filaments was observed in theisotype-control 1B7.11-treated group in comparison with the naive group,demonstrating the development of mechanical hypersensitivity. EE1antibody, when administered intravenously 22 hours after intraplantarCFA, was able to significantly decrease this FR at the different timesstudied compared with that obtained in the isotype-control1B7.11-treated group. (FIG. 10).

Reversal of mechanical hypersensitivity was 41±8% and 22±8% at D1, 36±9%and 32±9% at D4 and 27±10% and 50±9% at D7 for a 2.5 mg/kg and 30 mg/kgintravenous administration of EE1 antibody, respectively (Table 30).

TABLE 30 Effect of EE1 antibody on CFA-induced mechanicalhypersensitivity in male C57BI/6 mice Dose Day 1 post-CFA Day 4 post-CFADay 7 post-CFA (mg/kg, FR (%) FR (%) FR (%) Group i.v.) % effect %effect % effect Naive n.a. 41.3 ± 4.4 100 ± 11 43.8 ± 2.6    100 ± 8  45± 5 100 ± 13 Isotype- 30 81.3 ± 4.4  0 ± 11 78.8 ± 3     0 ± 8 82.5 ±2.5  0 ± 7 control 1B7.11 EE1 2.5  65 ± 3.3 41 ± 8 66.3 ± 3.2 (*) 36 ± 972.5 ± 3.7  27 ± 10 30 (**) 22 ± 8 67.5 ± 3.1 (*) 32 ± 9 (*) 50 ± 9 72.5± 3.1 63.8 ± 3.2 (*) (***) FR: Frequency of Response (in %) ± SEM, %effect ± SEM, n.a. not applicable (*) p < 0.05, (**) p < 0.01 and (***)p < 0.001, Two-Way ANOVA with time as repeated measure followed byDunnett's test for factor group for each level of factor time

B2. Thermal Hypersensitivity

For thermal hypersensitivity, measures of Paw Withdrawal Latencies (PWL,in seconds) in response to a radiant heat using a plantar apparatus(IITC, Woodland Hills, USA) were assessed.

To investigate the efficacy of EE1 antibody on pain-like behavior, wecalculated the reversal of thermal hypersensitivity (in %) as follows:

Percent reversals were calculated as(PWL_(postdose)−Meanisotype-control_(postdose))/(Mean isotype-control_(predose)−Meanisotype-control_(postdose))for each mouse.

Thermal hypersensitivities were not different between all groups atbaseline, before intraplantar injection of CFA (data not shown).

At D1, D4 and D7 after intraplantar injection of CFA, a significantdecrease in paw withdrawal latency of the injected paw was observed inisotype-control 1B7.11-treated group of mice, demonstrating that CFAinduced a thermal hypersensitivity (data not shown).

EE1 antibody, administered intravenously 22 hours after intraplantar CFAinjection (i.e. on Day 1 post-intraplantar CFA injection), and was notable to increase the Paw Withdrawal Latency at D1, whatever the dosetested (FIG. 11). However, EE1 significantly increased the PawWithdrawal Latency at D4 and this effect was also present at D7 (FIG.11).

Reversal of thermal hypersensitivity was 41±15% and 58±21% at D4 and46±10% and 52±17% at D7 for a 2.5 mg/kg and 30 mg/kg intravenousadministration of EE1, respectively (Table 31).

TABLE 31 Effect of EE1 antibody on CFA-induced thermal hypersensitivityin male C57BI/6 mice Dose Day 1 post-CFA Day 4 post-CFA Day 7 post-CFA(mg/kg, PWL (sec) PWL (sec) PWL (sec) Group i.v.) % effect % effect %effect Isotype- 30 3.3 ± 0.3 n.a. 4.1 ± 0.1 n.a. 3.6 ± 0.3    n.a.control 1B7.11 EE1 2.5 3.1 ± 0.3 −7 ± 10 5.2 ± 0.4 41 ± 15 5.0 ± 0.3 (*)46 ± 10 30 2.6 ± 0.2 −20 ± 5     5.7 ± 0.6 (*) 58 ± 21 5.2 ± 0.6 (*) 52± 17 PWL: Paw withdrawal latency ± SEM, % effect ± SEM, n.a. notapplicable (*) p < 0.05, Two-Way ANOVA with time as repeated measurefollowed by Dunnett's test for factor group for each level of factortime

C. CCI (Chronic Constriction Injury)-Induced Neuropathic-Like Pain(Bennett's Model)

CCI model was used as a model of peripheral nerve injury. Briefly, micewere anesthetized with Isoflurane (3%), and the right sciatic nerve wasexposed at mid thigh level through a small incision. Three looseligatures of 6.0 chromic gut (Ethicon) at 1 mm space were placed aroundthe sciatic nerve. The surgical procedure was completed by closing themuscles and skin. The day of CCI surgery was considered as Day 0. Groupsizes were 6-10 male C57BI/6 mice.

EE1 antibody was administered intravenously at 2.5 and 30 mg/kg on Day11 post surgery and mechanical and thermal hypersensitivities wereassessed on Day 12 (D12), Day 14 (D14) and Day 18 (D18) post-surgerywhich corresponded to Day 1 (D1), Day 3 (D3) and Day 7 (D7)post-treatment.

C1. Mechanical Hypersensitivity

Mechanical hypersensitivity was assessed by measuring hind pawwithdrawal thresholds (on both injured [i.e. Ipsi] and non-injured [i.e.Contra] paws) to an increasing pressure (in g) stimulus using a DynamicPlantar Aesthesiometer (Ugo-Basile, Italy); a steel rod was applied tothe hind paws of the mice with an increasing force (5 grams in 10seconds).

To investigate the efficacy of EE1 antibody on pain-like behavior, wedetermined the reversal of mechanical hypersensitivity as follows:percent reversals were calculatedas(Ipsi_(postdose)−Ipsi_(predose))/(Contra_(predose)−Ipsi_(predose))foreach mouse.

Following surgery, operated mice developed a robust sensitization tomechanical stimulus on the injured paw, whereas the non-injured paw wasnot affected. At Day 11, the mechanical sensitization on the injured pawreached a plateau (data not shown).

EE1 antibody, administered intravenously on Day 11 demonstrated a slighttendency to reverse CCI-induced mechanical hypersensitivity on D12, D14and D18 with 15.2±4.9% and 15.2±5.7% on D12, 26.8±5.7% and 25.7±4.5% onD14 and 30.3±7.1% and 20.8±5.9% on D18, at 2.5 and 30 mg/kg respectively(FIG. 12 and Table 32).

TABLE 32 Effect of EE1 antibody on CCI-induced mechanicalhypersensitivity in male C57BI/6 mice Dose Day 18 (mg/kg, Day 12post-CCI Day 14 post-CCI post-CCI % Group i.v.) % effect ± SEM % effect± SEM effect ± SEM Isotype- 30  0.2 ± 3.0  1.8 ± 4.3 18.1 ± 6.6 control1B7.11 EE1 2.5 15.2 ± 4.9 26.8 ± 5.7 (**) 30.3 ± 7.1 30 15.2 ± 5.7 25.7± 4.5 (*) 20.8 ± 5.9 (*), p < 0.05, and (**), p < 0.01 Two-Way ANOVAwith time as repeated measure followed by Dunnett's test for factorgroup for each level of factor time (statistics performed on Delta ipsivalues)

C2. Thermal Hypersensitivity

For thermal hypersensitivity, measures of Paw Withdrawal Latencies (inseconds) in response to a radiant heat using a plantar apparatus (IITC,Woodland Hills, USA) were assessed on the injected hind paw.

To investigate the efficacy of EE1 antibody on pain-like behavior, wecalculated the reversal of thermal hypersensitivity (in %) as follows:

Percent reversals were calculated as(Ipsi_(postdose)−Meanisotype-control_(postdose))/(Mean naive_(postdose)−Meanisotype-control_(postdose))for each mouse.

Following surgery, operated mice developed a robust sensitization tothermal stimulus on the injured paw, whereas the non-injured paw was notaffected. At Day 11, the thermal sensitization on the injured pawreached a plateau (data not shown).

EE1 antibody, administered intravenously on Day 11 did not significantlyincrease Paw Withdrawal Latency of the injured paw on D12, even if atrend was observed. However, from D14, EE1 antibody significantlyincreased the Paw Withdrawal (FIG. 13).

Reversal of thermal hypersensitivity was 41±16% and 56±24% at D12 and51±16% and 98±48% at D14 and 78±19% and 84±22% at D18, for a 2.5 mg/kgand 30 mg/kg intravenous administration of EE1 antibody, respectively(Table 33).

TABLE 33 Effect of EE1 antibody on CCI-induced thermal hypersensitivityin male C57BI/6 mice Kinetic evaluation Dose Day 12 post-CCI Day 14post-CCI Day 18 post-CCI (mg/kg, PWL (sec) PWL (sec) PWL (sec) Groupi.v.) % effect % effect % effect naive n.a. 6.3 ± 0.5 100 ± 19  6.2 ±0.4 100 ± 14  6.1 ± 0.5 100 ± 17  Isotype- 30 3.8 ± 0.2 0 ± 7 3.4 ± 0.20 ± 5 3.4 ± 0.2 0 ± 7 control 1B7.11 EE1 2.5 4.8 ± 0.4 41 ± 16 4.8 ± 0.551 ± 16    5.5 ± 0.5 (*) 78 ± 19 30 5.2 ± 0.6 56 ± 24    6.1 ± 1.3 (**)98 ± 48    5.7 ± 0.6 (**) 84 ± 22 PWL: Paw withdrawal latency ± SEM, %effect ± SEM, n.a. non applicable (*) p < 0.05, and (**) p < 0.01Two-Way ANOVA with time as repeated measure followed by Dunnett's testfor factor group for each level of factor time

1. An isolated monoclonal antibody or antigen binding fragment thereofthat: a) specifically binds to Kallidin or des-Arg₁₀-Kallidin but not toBradykinin or des-Arg₉-Bradykinin; b) specifically binds to Kallidin ordes-Arg₁₀-Kallidin with a KD of less than 1×10⁻¹⁰ M; c) specificallybinds to Kallidin or des-Arg₁₀-Kallidin with a K_(off) of less than1×10⁴ s⁻¹; and/or d) specifically binds to Kallidin ordes-Arg₁₀-Kallidin and inhibits binding to the bradykinin B1 receptor;e) specifically binds to the N-terminal Lysine residue of Kallidin ordes-Arg10-Kallidin; f) inhibits the binding of Kallidin ordes-Arg₁₀-Kallidin to a bradykinin-1 receptor; and/or g) specificallybinds to mouse Kallidin-like peptide (KLP). 2-4. (canceled)
 5. Theantibody or antigen binding fragment thereof of claim 1 comprising: i) aheavy chain variable domain comprising an HCDR3 amino acid sequenceselected from the group consisting of: a) SEQ ID NO: 7 [X₁Y X₂ X₃D X₄HAMX₅Y], wherein X₁ is Y, For H, X₂ is R, D, A, V, L, I, M, F, Y or W, X₃is Y, F, W or H, X₄ is D, E or Y, and, X₅ is D or E; b) SEQ ID NO: 63[X₁EYDGX₂YX₃X₄LDX₅], wherein X₁ is W or F, X₂ is N or no amino acid; X₃is Y or S, X₄ is D or P, and X₅ is F or Y; c) SEQ ID NO: 13; d) SEQ IDNO: 32; e) SEQ ID NO: 40; f) SEQ ID NO: 47; and g) SEQ ID NO: 55; ii) aheavy chain variable domain comprising an HCDR2 amino acid sequenceselected from the group consisting of: h) SEQ ID NO: 8[YFX₁PX₂NGNTGYNQKFRG], wherein X₁ is D, R, A, V, L, I, M, F, Y or W, andX₂ is Y, D, E, N, or Q; i) SEQ ID NO: 64 [WX₁DPENGDX₂X₃YAPKFQG], whereinX₁ is I, or V, X₂ is T, or S, and X₃ is G, or D; j) SEQ ID NO: 14 k) SEQID NO: 33; l) SEQ ID NO: 41; m) SEQ ID NO: 48; and n) SEQ ID NO: 56;iii) a heavy chain variable domain comprising an HCDR1 amino acidsequence selected from the group consisting of: o) SEQ ID NO: 9[GYSFTDYX₁IY], wherein X₁ is N, W or Y; p) SEQ ID NO: 65 [GFNIKDYYX₁H],wherein X₁ is L, or M; q) SEQ ID NO: 15; r) SEQ ID NO: 34; s) SEQ ID NO:42; t) SEQ ID NO: 49; and u) SEQ ID NO: 57; iv) a light chain variabledomain comprising an LCDR3 amino acid sequence selected from the groupconsisting of: v) SEQ ID NO: 10 [QQ X₁ X₂S X₃P X₄T], wherein X₁ is Y,For H, X₂is Y, F, H or W, X₃ is Y, F, T or H, and, X₄ is W, Y, F, H orL: w) SEQ ID NO: 66 [QX₁X₂X₃SX₄PX₅T], wherein X₁ is Q or N, X₂is Y, F, Dor H, X₃ is Y, F, H or W, X₄ is Y, F, T or H, and X₅ is W, Y, F, H or L;x) SEQ ID NO: 69 [X₁QGTHFPYT], wherein X₁ is L or M; z) SEQ ID NO: 16;aa) SEQ ID NO: 35; bb) SEQ ID NO: 43; cc) SEQ ID NO: 50; and dd) SEQ IDNO: 58; vi) a light chain variable domain comprising an LCDR2 amino acidsequence selected from the group consisting of: ee) SEQ ID NO: 11[WASTRX₁], wherein X₁ is E, D, Q or N; ff) SEQ ID NO: 67 [X₂ASTRX₂],wherein X₁ is W or G, and X₂ is E, D, Q or N; gg) SEQ ID NO: 17; hh) IDNO: 36; ii SE ID NO: 51; and jj) SEQ ID NO: 59; and/or vii) a lightchain variable domain comprising an LCDR1 amino acid sequence selectedfrom the group consisting of: kk) SEQ ID NO: 12 [KSSQSLL X₁SSNQKN X₂LA],wherein X₁ is W, H, Y or F, and X₂ is H or Y; ll) SEQ ID NO: 68[KSSQSLLX₁X₂SX₃QX₄NX₅LA], wherein X₁ is W, H, Y or F, X₂ is S or G, X₃is N or D, X₄ is K or R, X₅ is H or Y. mm) SEQ ID NO: 70[KSSQSLLYSNGX1TYLN], wherein X1 is K or E; nn) SEQ ID NO: 18; oo) SEQ IDNO: 37; pp) SEQ ID NO: 44; qq) SEQ ID NO: 52; and rr) SEQ ID NO: 60.6-13. (canceled)
 14. The antibody or antigen binding fragment thereof ofclaim 1 comprising: a) a heavy chain variable region comprising theHCDR3, HCDR2 and HCDR1 region amino sequences set forth in SEQ ID NOs13, 14, and 15, respectively, and one or more amino acid substitution atpositions selected from the group consisting of H1, H5, H9, H11, H12,H16, H38, H40, H41, H43, H44, H66, H75, H79, H81, H82A, H83, H87, andH108 according to Kabat; b) a light chain variable region comprising theLCDR3, LCDR2 and LCDR1 region amino sequences set forth in SEQ ID NOs16, 17, and 18, respectively, and one or more amino acid substitution atpositions selected from the group consisting of L5, L9, L15 L18, L19,L21, L22, L43, L63, L78, L79, L83, L85, L100 and L104, according toKabat; c) a light chain variable region comprising the LCDR3, LCDR2 andLCDR1 region amino sequences set forth in SEQ ID NOs 16, 17, and 18,respectively, and one or more amino acid substitution at positionsselected from the group consisting of L5, L9, L15, L18, L19, L21, L22,L43, L63, L78, L79, L83, L85, L100 and L104 according to Kabat; d) aheavy chain variable region amino acid sequence with at least 90%identity to an amino acid sequence selected from the group consistingSEQ ID NOs: 19, 20, 21, 22, 24, 25, 38, 45, 53, and 61; e) a light chainvariable domain amino acid sequence with at least 90% identity to anamino acid sequence selected from the group consisting SEQ ID NOs: 26,27, 28, 29, 29, 30, 31, 39, 46, 54, and 62; f) a light chain variableregion amino acid sequence with at least 90% identity to an amino acidsequence selected from the group consisting of SEQ ID NOs: 26, 27, 28,29, 29, 30, 31, 39, 46, 54, and 62; g) a heavy chain variable domaincomprising an amino acid sequence selected from the group consisting of:SEQ ID NO: 19, 20, 21, 22, 24, 25, 38, 45, 53, and 61; and h) a lightchain variable domain amino acid sequence selected from the groupconsisting of: SEQ ID NO: 26, 27, 28, 29, 29, 30, 31, 39, 46, 54, and62; and/or i) an amino acid sequence selected from the group consistingof: SEQ ID NO: 26, 27, 28 29, 29, 30, 31, 39, 46, 54, and
 62. 15-22.(canceled)
 23. The antibody or antigen binding fragment thereof of claim1 comprising the heavy chain and light chain variable region amino acidsequences set forth in SEQ ID NO: 24 and 30, respectively.
 24. Anantibody, or antigen binding fragment thereof, that specifically bindsto Kallidin and des-Arg₁₀-Kallidin, wherein the antibody, or antigenbinding fragment thereof, competes for binding to Kallidin anddes-Arg₁₀-Kallidin with the antibody or antigen binding fragment ofclaim
 23. 25. An isolated monoclonal antibody or antigen bindingfragment thereof that competes for binding to Kallidin ordes-Arg₁₀-Kallidin with the antibody or antigen binding fragment thereofof claim 14, and does not bind to Bradykinin or desArg₉-Bradykinin. 26.The isolated monoclonal antibody or antigen binding fragment thereof ofclaim 1 wherein the antibody further specifically binds to aconformational epitope of kallidin (KD) or desArg10-Kallidin (DAKD)which adopts a Pro4 kink conformation comprising a type II tight turn atProline 4 of the KD or DAKD.
 27. The antibody or antigen bindingfragment of claim 26, wherein the Pro 4 kink conformation of KD or DAKDfurther comprises amino acid repeats of a sigmoid shape which align thehydrophobic side chains of the amino acids in a spatially stacking mode.28. (canceled)
 29. The antibody or antigen binding fragment thereof ofclaim 1 conjugated to a diagnostic or therapeutic agent.
 30. Theisolated nucleic acid encoding the amino acid sequence of the antibody,or antigen binding fragment thereof, of claim
 1. 31. A recombinantexpression vector comprising the nucleic acid of claim
 30. 32. A hostcell comprising the recombinant expression vector of claim
 31. 33. Amethod of producing an antibody that binds specifically to Kallidin anddes-Arg₁₀-Kallidin, comprising culturing the host cell of claim 32 underconditions such that an antibody that binds specifically to Kallidin anddes-Arg₁₀-Kallidin is produced by the host cell.
 34. A pharmaceuticalcomposition comprising the antibody, or antigen binding fragmentthereof, of claim 1 and one or more pharmaceutically acceptablecarrier(s).
 35. A method for use of the antibody or antigen bindingfragment thereof of claim 1 to manufacture a medicament for thetreatment of a Kallidin or des-Arg₁₀-Kallidin-associated disease ordisorder.
 36. The method of claim 35, wherein the disease or disorder ischronic pain.
 37. A method of generating the antibody or antigen bindingfragment thereof of claim 1 that specifically binds todes-Arg₉-Bradykinin and des-Arg₁₀-Kallidin-like peptide comprising:immunizing an animal with an immunogen comprising a peptide, wherein thepeptide consists of the amino acid sequence set forth in SEQ ID No. 11,and wherein the amino terminal arginine of the peptide is indirectlycoupled to a carrier moiety through a linker moiety, such that anantibody that specifically binds to des-Arg₉-Bradykinin,des-Arg₁₀-Kallidin and des-Arg₁₀-Kallidin-like peptide is produced bythe immune system of the animal.
 38. The method of claim 37, furthercomprising isolating from the animal, the antibody, a nucleic isolatingencoding the antibody, or an immune cell expressing the antibody. 39.The method of claim 37, wherein the carrier moiety is a protein. 40.(canceled)
 41. The method of claim 37, wherein the linker moietycomprises [Gly-Gly-Gly]n, wherein n is at least 1.